Processes for recovering oil from ethanol production processes

ABSTRACT

The present invention relates to processes for recovering/extracting oil from fermentation product production processes based on starch-containing material, wherein an alpha-amylase, a high dosage of protease, and optionally a glucoamylase, are present and/or added in liquefaction. The invention also relates to processes for producing fermentation products and to enzyme compositions suitable for use in processes of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 14/901,508filed Dec. 28, 2015, now allowed, which is a 35 U.S.C. 371 nationalapplication of PCT/US2014/043444 filed Jun. 20, 2014, which claimspriority or the benefit under 35 U.S.C. 119 of U.S. provisionalapplication Nos. 61/838,650, 61/863,727, 61/943,794 and 61/991,866 filedJun. 24, 2013, Aug. 8, 2013, Feb. 24, 2014 and May 12, 2014,respectively, the contents of which are fully incorporated herein byreference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form.The computer readable form is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to processes of recovering oil from afermentation product production process and well as processes forproducing fermentation products from starch-containing material. Theinvention also relates to compositions suitable for use in a process ofthe invention.

BACKGROUND OF THE INVENTION

Fermentation products, such as ethanol, are typically produced by firstgrinding starch-containing material in a dry-grind or wet-millingprocess, then degrading the material into fermentable sugars usingenzymes and finally converting the sugars directly or indirectly intothe desired fermentation product using a fermenting organism. Liquidfermentation products are recovered from the fermented mash (oftenreferred to as “beer mash”), e.g., by distillation, which separate thedesired fermentation product from other liquids and/or solids. Theremaining faction is referred to as “whole stillage”. The whole stillageis dewatered and separated into a solid and a liquid phase, e.g., bycentrifugation. The solid phase is referred to as “wet cake” (or “wetgrains”) and the liquid phase (supernatant) is referred to as “thinstillage”. Wet cake and thin stillage contain about 35 and 7% solids,respectively. Dewatered wet cake is dried to provide “Distillers DriedGrains” (DDG) used as nutrient in animal feed. Thin stillage istypically evaporated to provide condensate and syrup or mayalternatively be recycled directly to the slurry tank as “backset”.Condensate may either be forwarded to a methanator before beingdischarged or may be recycled to the slurry tank. The syrup may beblended into DDG or added to the wet cake before drying to produce DDGS(Distillers Dried Grain with Solubles).

WO 2012/088303 (Novozymes) discloses processes for producingfermentation products by liquefying starch-containing material at a pHin the range from 4.5-5.0 at a temperature in the range from 80-90° C.using a combination of alpha-amylase having a T½ (min) at pH 4.5, 85°C., 0.12 mM CaCl2)) of at least 10 and a protease having athermostability value of more than 20% determined as Relative Activityat 80° C./70° C.; followed by saccharification and fermentation.

WO 2013/082486 (Novozymes) discloses processes for producingfermentation products by liquefying starch-containing material at a pHin the range between from above 5.0-7.0 at a temperature above theinitial gelatinization temperature using an alpha-amylase; a proteasehaving a thermostability value of more than 20% determined as RelativeActivity at 80° C./70° C.; and optionally a carbohydrate-sourcegenerating enzyme followed by saccharification and fermentation.

An increasing number of ethanol plants extract oil from the thinstillage and/or syrup as a by-product for use in biodiesel production orother biorenewable products. Much of the work in oil recovery/extractionfrom fermentation product production processes has focused on improvingthe extractability of the oil from the thin stillage. Effective removalof oil is often accomplished by hexane extraction. However, theutilization of hexane extraction has not seen widespread application dueto the high capital investment required. Therefore, other processes thatimprove oil extraction from fermentation product production processeshave been explored.

WO 2011/126897 (Novozymes) discloses processes of recovering oil byconverting starch-containing materials into dextrins with alpha-amylase;saccharifying with a carbohydrate source generating enzyme to formsugars; fermenting the sugars using fermenting organism; wherein thefermentation medium comprises a hemicellulase; distilling thefermentation product to form whole stillage; separating the wholestillage into thin stillage and wet cake; and recovering oil from thethin stillage. The fermentation medium may further comprise a protease.

It is an object of the present invention to provide improved processesfor increasing the amount of recoverable oil from fermentation productproduction processes and to provide processes for producing fermentationproducts, such as ethanol, from starch-containing material that canprovide a higher fermentation product yield, or other advantages,compared to a conventional process.

SUMMARY OF THE INVENTION

The present invention relates to processes of recovering/extracting oilfrom fermentation product production processes. The invention alsorelated to producing fermentation products, such as ethanol, fromstarch-containing material in a process including liquefyingstarch-containing material, saccharifying and fermenting the liquefiedmaterial. The invention also relates to compositions suitable for use ina process of the invention.

In the first aspect the invention relates to processes ofrecovering/extracting oil from a fermentation product production processcomprising the steps of:

-   -   a) liquefying starch-containing material at a temperature above        the initial gelatinization temperature using:        -   an alpha-amylase;        -   more than 0.5 micro gram Pyrococcus furiosus protease per            gram dry solids (DS);    -   b) saccharifying using a glucoamylase;    -   c) fermenting using a fermenting organism.    -   d) recovering the fermentation product to form whole stillage;    -   e) separating the whole stillage into thin stillage and wet        cake;    -   f) optionally concentrating the thin stillage into syrup;

wherein oil is recovered from the:

-   -   liquefied starch-containing material during and/or after step        a); and/or    -   downstream from fermentation step c).

In an embodiment between 0.5-100 micro gram Pyrococcus furiosus proteaseper gram DS (dry solids) DS is present and/or added in liquefaction stepa). In an embodiment between 0.5-10 micro gram Pyrococcus furiosusprotease per gram DS (dry solids) is present and/or added inliquefaction step a). In an embodiment between 1-50 micro gramPyrococcus furiosus protease per gram DS is present and/or added inliquefaction step a). In an embodiment between 1-10 micro gramPyrococcus furiosus protease per gram DS is present and/or added inliquefaction step a). In an embodiment between 1.5-5 micro gramPyrococcus furiosus protease per gram DS is present and/or added inliquefaction step a). In an embodiment around or more than 1.5 microgram Pyrococcus furiosus protease per gram DS is present and/or added inliquefaction step a). In an embodiment around or more than 2 micro gramPyrococcus furiosus protease per gram DS is present and/or added inliquefaction step a). In an embodiment around or more than 3 micro gramPyrococcus furiosus protease per gram DS is present and/or added inliquefaction step a).

In a preferred embodiment the Pyrococcus furiosus protease is the maturesequence shown in SEQ ID NO: 13 herein or one having at least 90% or 95%identity thereof.

Examples of alpha-amylase can be found below in the “Alpha-AmylasesPresent and/or Added In Liquefaction”-section below.

Preferred alpha-amylases are Bacillus sp. alpha-amylases or variantsthereof, especially derived from Bacillus stearothermophilus or Bacilluslicheniformis.

In a preferred embodiment the alpha-amylase is a Bacillusstearothermophilus alpha-amylase variant comprising a double deletion inpositions I181*+G182* (using SEQ ID NO: 1 for numbering).

Preferred alpha-amylases include Bacillus stearothermophilusalpha-amylase variants, such as one shown in SEQ ID NO: 1 herein withthe following mutations:

-   -   I181*+G182*+N193F+E129V+K177L+R179E;    -   I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S    -   I181*+G182*+N193F+V59A Q89R+E129V+K177L+R179E+Q254S+M284V; and    -   I181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S        (using SEQ ID NO: 1 for numbering).

In an embodiment a glucoamylase is present and/or added in liquefaction.Examples of suitable glucoamylase can be found in the “GlucoamylasePresent And/Or Added In Liquefaction” section below.

In an embodiment the glucoamylase has a thermostability of at least 80°C., preferably at least 82° C., such as at least 84° C., such as atleast 86° C., such as at least 88° C. at pH 4.0 determined asDifferential Scanning Calorimitry (DSC) as described in Example 3 below.

In an embodiment the glucoamylase has a thermostability of at least 80°C., preferably at least 82° C., such as at least 84° C., such as atleast 86° C., such as at least 88° C., such as at least 90° C. at pH 4.8determined as Differential Scanning Calorimitry (DSC) as described inExample 3 below.

Examples of specifically contemplated glucoamylases can be found inExample 3 (Table 6) below.

Preferred glucoamylases include Penicillium oxalicum glucoamylases, suchas one shown in SEQ ID NO: 14 herein having a K79V substitution andpreferably further one of the following:

-   -   P11F+T65A+Q327F;    -   P2N+P4S+P11F+T65A+Q327F (using SEQ ID NO: 14 for numbering).

In a preferred embodiment liquefaction is carried out at a temperaturebetween 80-90° C., such as around 85° C. In a preferred embodimentliquefaction is carried out at a pH in the range pH above 5.0 to 6.0.

A glucoamylase is present and/or added in saccharification and/orfermentation. Examples of suitable glucoamylases can be found in the“Glucoamylase Present And/Or Added In Saccharification And/OrFermentation” section below.

In a second aspect the invention relates to processes for producingfermentation products from starch-containing material comprising thesteps of:

-   -   a) liquefying the starch-containing material at a temperature        above the initial gelatinization temperature using:        -   an alpha-amylase;        -   more than 2 micro gram Pyrococcus furiosus protease per gram            dry solids (DS);    -   b) saccharifying using a glucoamylase;    -   c) fermenting using a fermenting organism.

In a preferred embodiment between 2-100 micro gram Pyrococcus furiosusprotease per gram DS is present and/or added in liquefaction step a). Ina preferred embodiment between 2-10 micro gram Pyrococcus furiosusprotease per gram DS is present and/or added in liquefaction step a). Ina preferred embodiment between 2.5-50 micro gram Pyrococcus furiosusprotease per gram DS is present and/or added in liquefaction step a). Ina preferred embodiment between 2.5-10 micro gram per gram DS is presentand/or added in liquefaction step a). In a preferred embodiment between2.5-5 micro gram Pyrococcus furiosus protease per gram DS is presentand/or added in liquefaction step a). In a preferred embodiment aroundor more than 3 micro gram Pyrococcus furiosus protease per gram DS ispresent and/or added in liquefaction step a).

In a preferred embodiment the Pyrococcus furiosus protease is the matureone shown in SEQ ID NO: 13 herein or one having at least 90% or at least95% identity thereof.

Examples of alpha-amylase can be found below in the “Alpha-AmylasesPresent and/or Added In Liquefaction”-section below.

Preferred alpha-amylases are Bacillus sp. alpha-amylases or variantsthereof, especially derived from Bacillus stearothermophilus or Bacilluslicheniformis.

In a preferred embodiment the alpha-amylase is a Bacillusstearothermophilus alpha-amylase variant comprising a double deletion inI181*+G182* (using SEQ ID NO: 1 for numbering).

Preferred alpha-amylases include Bacillus stearothermophilusalpha-amylase variants, such as one shown in SEQ ID NO: 1 herein withthe following mutations:

-   -   I181*+G182*+N193F+E129V+K177L+R179E;    -   I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S    -   I181*+G182*+N193F+V59A Q89R+E129V+K177L+R179E+Q254S+M284V; and    -   I181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S        (using SEQ ID NO: 1 for numbering).

In an embodiment glucoamylase is present and/or added in liquefaction.Examples of suitable glucoamylase can be found in the “GlucoamylasePresent And/Or Added In Liquefaction” section below.

In an embodiment the glucoamylase has a thermostability of at least 80°C., preferably at least 82° C., such as at least 84° C., such as atleast 86° C., such as at least 88° C. at pH 4.0 determined asDifferential Scanning Calorimitry (DSC) as described in Example 3 below.

In an embodiment the glucoamylase has a thermostability of at least 80°C., preferably at least 82° C., such as at least 84° C., such as atleast 86° C., such as at least 88° C., such as at least 90° C. at pH 4.8determined as Differential Scanning Calorimitry (DSC) as described inExample 3 below.

Examples of specifically contemplated glucoamylases can be found inExample 3 (Table 6) below.

Preferred glucoamylases include Penicillium oxalicum glucoamylases, suchas one shown in SEQ ID NO: 14 herein having a K79V substitution andpreferably further one of the following:

-   -   P11F+T65A+Q327F;    -   P2N+P4S+P11F+T65A+Q327F (using SEQ ID NO: 14 for numbering).

A glucoamylase is present and/or added in saccharification and/orfermentation. Examples of suitable glucoamylases can be found in the“Glucoamylase Present And/Or Added In Saccharification And/OrFermentation” section below.

In a third aspect the invention relates to an enzyme compositioncomprising:

-   -   (i) Bacillus sp. alpha-amylase, or a variant thereof;    -   (ii) Pyrococcus furiosus protease;

wherein the ratio between alpha-amylase and protease is in the rangefrom 1:1 and 1:25 (micro gram alpha-amylase:micro gram protease).

Examples of alpha-amylase can be found below in the “Alpha-AmylasesPresent And/Or Added In Liquefaction” section below.

Preferred alpha-amylases are Bacillus sp. alpha-amylases or variantsthereof, especially derived from Bacillus stearothermophilus or Bacilluslicheniformis.

In a preferred embodiment the alpha-amylase is a Bacillusstearothermophilus alpha-amylase variant comprising a double deletion inI181*+G182* (using SEQ ID NO: 1 for numbering).

Preferred alpha-amylases include Bacillus stearothermophilusalpha-amylase variants, such as one show in SEQ ID NO: 1 herein with thefollowing mutations:

-   -   I181*+G182*+N193F+E129V+K177L+R179E;    -   I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S    -   I181*+G182*+N193F+V59A Q89R+E129V+K177L+R179E+Q254S+M284V; and    -   I181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S        (using SEQ ID NO: 1 for numbering).

In an embodiment a glucoamylase is present and/or added in liquefaction.Examples of suitable glucoamylase can be found in the “GlucoamylasePresent And/Or Added In Liquefaction” section below.

In an embodiment the glucoamylase has a thermostability of at least 80°C., preferably at least 82° C., such as at least 84° C., such as atleast 86° C., such as at least 88° C. at pH 4.0 determined asDifferential Scanning Calorimitry (DSC) as described in Example 3 below.

In an embodiment the glucoamylase has a thermostability of at least 80°C., preferably at least 82° C., such as at least 84° C., such as atleast 86° C., such as at least 88° C., such as at least 90° C. at pH 4.8determined as Differential Scanning Calorimitry (DSC) as described inExample 3 below.

Examples of specifically contemplated glucoamylases can be found inExample 3 (Table 6) below.

Preferred glucoamylases include Penicillium oxalicum glucoamylases, suchas one shown in SEQ ID NO: 14 herein having a K79V substitution andfurther one of the following:

-   -   P11F+T65A+Q327F;    -   P2N+P4S+P11F+T65A+Q327F (using SEQ ID NO: 14 for numbering).

In a preferred embodiment the ratio between alpha-amylase and proteaseis in the range between 1:1.2 and 1:10, such as around 1:1.4 (micro gramalpha-amylase:micro gram protease).

In another embodiment the enzyme composition of the invention comprisesa glucoamylase and the ratio between alpha-amylase and glucoamylase inliquefaction is between 1:1 and 1:10, such as around 1:2 (micro gramalpha-amylase:micro gram glucoamylase).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an oil extraction comparison between Protease Pfu (1.5μg/gDS) and Protease X (no statistical difference).

FIG. 2 shows an oil extraction comparison between Protease Pfu (3μg/gDS) and Protease X (statistical difference).

FIG. 3 shows an oil extraction comparison between Protease Pfu (5μg/gDS) and Protease X (statistical difference).

FIG. 4 shows the ethanol concentrations (% w/v) for no-ureafermentations.

FIG. 5 shows an Oneway Analysis of the ethanol concentration (% w/v)comparison for 0, 1.5, 3 and 5 μg/gDS Protease Pfu comparison urea-freefermentations.

FIG. 6 shows the ethanol concentrations (% w/v) for fermentationsoperating with 200 ppm urea for Protease X added in SSF and Protease Pfu(1.5, 3 and 5 μg/gDS.

FIG. 7 shows an Oneway Analysis of the ethanol concentration (% w/v)comparison for 0, 1.5, 3 and 5 μg/gDS Protease Pfu for 200 ppm ureabased fermentations.

FIG. 8 shows the 54 hour glycerol concentrations (% w/v). The highestdose of Protease Pfu (5 μg/gDS) was approximately 10% lower than thecontrol of Protease X.

FIG. 9 shows the ethanol concentrations (% w/v) after 54 hours when from0 (control) to 50 μg/gDS Protease Pfu was added in liquefaction.

FIG. 10 shows the glycerol concentrations (% w/v) after 54 hours whenfrom 0 (control) to 50 μg/gDS Protease Pfu was added in liquefaction.

FIG. 11 shows the glucose concentrations (% w/v) after 54 hours whenfrom 0 (control) to 50 μg/gDS Protease Pfu was added in liquefaction.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to processes of recovering oil from afermentation product production process and well as processes forproducing fermentation products from starch-containing material. Theinvention also relates to compositions suitable for use in a process ofthe invention.

The inventors have found that an increased amount of oil can berecovered in liquefaction or downstream from fermentation when combiningan alpha-amylase, a high amount of Pyrococcus furiosus protease andoptionally a glucoamylase compared to when adding an alpha-amylase inliquefaction and a protease during fermentation (SSF).

The inventors also found that an increased ethanol yield is obtainedwhen combining an alpha-amylase, more than 2 micro gram Pyrococcusfuriosus protease per gram dry solids (DS) and a glucoamylase comparedto when using alpha-amylase, less than 2 micro gram Pyrococcus furiosusprotease per gram dry solids (DS) and glucoamylase during liquefaction.

The inventors also found that the glycerol concentration is lower withProtease Pfu (5 μg/gDS) compared to adding protease in SSF.

It was also found that an ethanol process of the invention can be runefficiently with reduced or without adding a nitrogen source, such asurea, in SSF.

Processes of Recovering/Extracting Oil of the Invention

In the first aspect the invention relates to processes of recovering oilfrom a fermentation product production process comprising the steps of:

-   -   a) liquefying starch-containing material at a temperature above        the initial gelatinization temperature using:        -   an alpha-amylase;        -   more than 0.5 micro gram Pyrococcus furiosus protease per            gram dry solids (DS);    -   b) saccharifying using a glucoamylase;    -   c) fermenting using a fermenting organism.    -   d) recovering the fermentation product to form whole stillage;    -   e) separating the whole stillage into thin stillage and wet        cake;    -   f) optionally concentrating the thin stillage into syrup;

wherein oil is recovered from the:

-   -   liquefied starch-containing material after step a); and/or    -   downstream from fermentation step c).

In an embodiment the oil is recovered/extracted during and/or afterliquefying the starch-containing material. In an embodiment the oil isrecovered from the whole stillage. In an embodiment the oil is recoveredfrom the thin stillage. In an embodiment the oil is recovered from thesyrup.

In an embodiment between 0.5-100 micro gram Pyrococcus furiosus proteaseper gram DS (dry solids) DS is present and/or added in liquefaction stepa). In an embodiment between 0.5-10 micro gram Pyrococcus furiosusprotease per gram DS (dry solids) DS is present and/or added inliquefaction step a). In an embodiment between 1-50 micro gramPyrococcus furiosus protease per gram DS is present and/or added inliquefaction step a). In an embodiment between 1-10 micro gramPyrococcus furiosus protease per gram DS is present and/or added inliquefaction step a). In an embodiment between 1.5-5 micro gramPyrococcus furiosus protease per gram DS is present and/or added inliquefaction step a). In an embodiment around or more than 1 micro gramPyrococcus furiosus protease per gram DS is present and/or added inliquefaction step a). In an embodiment around or more than 1.5 microgram Pyrococcus furiosus protease per gram DS is present and/or added inliquefaction step a). In an embodiment around or more than 2 micro gramPyrococcus furiosus protease per gram DS is present and/or added inliquefaction step a).

In an embodiment between 2-100 micro gram Pyrococcus furiosus proteaseper gram DS is added and/or present during liquefaction. In anembodiment between 2.5-50 micro gram Pyrococcus furiosus protease pergram DS is added and/or present during liquefaction. In an embodimentbetween 2.5-10 micro gram Pyrococcus furiosus protease per gram DS isadded and/or present during liquefaction. In an embodiment between 2.5-5micro gram Pyrococcus furiosus protease per gram DS is added and/orpresent during liquefaction. In an embodiment between 2.75-50 micro gramPyrococcus furiosus protease per gram DS is added and/or present duringliquefaction. In an embodiment between 2.75-10 micro gram Pyrococcusfuriosus protease per gram DS is added and/or present duringliquefaction. In an embodiment between 2.75-5 micro gram Pyrococcusfuriosus protease per gram DS is added and/or present duringliquefaction. In a preferred embodiment around or more than 3 micro gramPyrococcus furiosus protease per gram DS are present and/or added inliquefaction step a).

In a preferred embodiment the Pyrococcus furiosus protease is the maturesequence shown in SEQ ID NO: 13 herein. In an embodiment the Pyrococcusfuriosus protease is one having at least 80%, such as at least 85%, suchas at least 90%, such as at least 95%, such as at least 96%, such as atleast 97%, such as at least 98%, such as at least 99% identity to SEQ IDNO: 13 herein.

In an embodiment no nitrogen-compound, such as urea, is present and/oradded in steps a)-c), such as during saccharification step b),fermentation step c), or simultaneous saccharification and fermentation(SSF).

In an embodiment 10-1,000 ppm, such as 50-800 ppm, such as 100-600 ppm,such as 200-500 ppm nitrogen-compound, preferably urea, is presentand/or added in steps a)-c), such as in saccharification step b) orfermentation step c) or in simultaneous saccharification andfermentation (SSF).

Process of Producing a Fermentation Product of the Invention

In the second aspect the invention relates to processes for producingfermentation products from starch-containing material comprising thesteps of:

-   -   a) liquefying the starch-containing material at a temperature        above the initial gelatinization temperature using:        -   an alpha-amylase;        -   more than 2 micro gram Pyrococcus furiosus protease per gram            dry solids (DS);    -   b) saccharifying using a glucoamylase;    -   c) fermenting using a fermenting organism.

In an embodiment the fermentation product is recovered afterfermentation. In a preferred embodiment the fermentation product isrecovered after fermentation, such as by distillation. In an embodimentthe fermentation product is an alcohol, preferably ethanol, especiallyfuel ethanol, potable ethanol and/or industrial ethanol.

In an embodiment from 2-100 micro gram Pyrococcus furiosus protease pergram DS is added and/or present during liquefaction. In an embodiment2.5-50 micro gram Pyrococcus furiosus protease per gram DS is addedand/or present during liquefaction. In an embodiment 2.5-10 micro gramPyrococcus furiosus protease per gram DS is added and/or present duringliquefaction. In an embodiment 2.5-5 micro gram Pyrococcus furiosusprotease per gram DS is added and/or present during liquefaction. In anembodiment 2.75-50 micro gram Pyrococcus furiosus protease per gram DSis added and/or present during liquefaction. In an embodiment 2.75-10micro gram Pyrococcus furiosus protease per gram DS is added and/orpresent during liquefaction. In an embodiment 2.75-5 micro gramPyrococcus furiosus protease per gram DS is added and/or present duringliquefaction. In a preferred embodiment around 3 micro gram Pyrococcusfuriosus protease per gram DS is present and/or added in liquefactionstep a).

In a preferred embodiment the Pyrococcus furiosus protease is the oneshown in SEQ ID NO: 13 herein. In an embodiment the Pyrococcus furiosusprotease is one having at least 80%, such as at least 85%, such as atleast 90%, such as at least 95%, such as at least 96%, such as at least97%, such as at least 98%, such as at least 99% identity to SEQ ID NO:13 herein.

In a preferred embodiment no nitrogen-compound is present and/or addedin steps a)-c), such as during saccharification step b) or fermentationstep c) or simultaneous saccharification and fermentation (SSF).

In an embodiment 10-1,000 ppm, such as 50-800 ppm, such as 100-600 ppm,such as 200-500 ppm nitrogen-compound, preferably urea, is presentand/or added in steps a)-c), such as during saccharification step b) orfermentation step c) or simultaneous saccharification and fermentation(SSF).

Alpha-Amylases Present and/or Added in Liquefaction

The alpha-amylase added during liquefaction step a) in a process of theinvention (i.e., oil recovery process and fermentation productproduction process) may be any alpha-amylase.

Preferred are bacterial alpha-amylases, which typically are stable at atemperature used in liquefaction.

In an embodiment the alpha-amylase is from a strain of the genusBacillus.

In a preferred embodiment the alpha-amylase is from a strain of Bacillusstearothermophilus, such as the sequence shown in SEQ ID NO: 1. In anembodiment the alpha-amylase is the Bacillus stearothermophilusalpha-amylase shown in SEQ ID NO: 1 herein, such as one having at least80%, such as at least 85%, such as at least 90%, such as at least 95%,such as at least 96%, such as at least 97%, such as at least 98%, suchas at least 99% identity to SEQ ID NO: 1 herein.

In an embodiment the Bacillus stearothermophilus alpha-amylase orvariant thereof is truncated, preferably at the C-terminal, preferablytruncated to have around 491 amino acids, such as from 480-495 aminoacids.

In an embodiment the Bacillus stearothermophilus alpha-amylase has adouble deletion at positions I181+G182, and optionally a N193Fsubstitution (using SEQ ID NO: 1 for numbering).

In another embodiment the Bacillus stearothermophilus alpha-amylase hasa double deletion at positions R179+G180 and optionally a N193Fsubstitution (using SEQ ID NO: 1 for numbering).

In an embodiment the Bacillus stearothermophilus alpha-amylase has asubstitution at position S242, preferably S242Q substitution.

In an embodiment the Bacillus stearothermophilus alpha-amylase has asubstitution at position E188, preferably E188P substitution.

In an embodiment the alpha-amylase has a T½ (min) at pH 4.5, 85° C.,0.12 mM CaCl₂) of at least 10.

In embodiment the alpha-amylase has a T½ (min) at pH 4.5, 85° C., 0.12mM CaCl₂) of at least 15.

In embodiment the alpha-amylase has a T½ (min) at pH 4.5, 85° C., 0.12mM CaCl₂) of at least 20.

In embodiment the alpha-amylase has a T½ (min) at pH 4.5, 85° C., 0.12mM CaCl₂) of at least 25.

In embodiment the alpha-amylase has a T½ (min) at pH 4.5, 85° C., 0.12mM CaCl₂) of at least 30.

In embodiment the alpha-amylase has a T½ (min) at pH 4.5, 85° C., 0.12mM CaCl₂) of at least 40.

In embodiment the alpha-amylase has a T½ (min) at pH 4.5, 85° C., 0.12mM CaCl₂) of at least 50.

In embodiment the alpha-amylase has a T½ (min) at pH 4.5, 85° C., 0.12mM CaCl₂) of at least 60. In embodiment the alpha-amylase has a T½ (min)at pH 4.5, 85° C., 0.12 mM CaCl₂) between 10-70.

In embodiment the alpha-amylase has a T½ (min) at pH 4.5, 85° C., 0.12mM CaCl₂) between 15-70.

In embodiment the alpha-amylase has a T½ (min) at pH 4.5, 85° C., 0.12mM CaCl₂) between 20-70.

In embodiment the alpha-amylase has a T½ (min) at pH 4.5, 85° C., 0.12mM CaCl₂) between 25-70.

In embodiment the alpha-amylase has a T½ (min) at pH 4.5, 85° C., 0.12mM CaCl₂) between 30-70.

In embodiment the alpha-amylase has a T½ (min) at pH 4.5, 85° C., 0.12mM CaCl₂) between 40-70.

In embodiment the alpha-amylase has a T½ (min) at pH 4.5, 85° C., 0.12mM CaCl₂) between 50-70.

In embodiment the alpha-amylase has a T½ (min) at pH 4.5, 85° C., 0.12mM CaCl₂) between 60-70.

In an embodiment the alpha-amylase is selected from the group ofBacillus stearothermophilus alpha-amylase variants with the followingmutations in addition to I181*+G182*, and optionally N193F:

-   -   V59A+Q89R+G112D+E129V+K177L+R179E+K220P+N224L+Q254S;    -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;    -   V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+D269E+D281N;    -   V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+1270L;    -   V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+H274K;    -   V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+Y276F;    -   V59A+E129V+R157Y+K177L+R179E+K220P+N224L+S242Q+Q254S;    -   V59A+E129V+K177L+R179E+H208Y+K220P+N224L+S242Q+Q254S;    -   59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+H274K;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+Y276F;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+D281N;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+M284T;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+G416V;    -   V59A+E129V+K177L+R179E+K220P+N224L+Q254S;    -   V59A+E129V+K177L+R179E+K220P+N224L+Q254S+M284T;    -   A91L+M961+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;    -   E129V+K177L+R179E;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+Y276F+L427M;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+M284T;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+N376*+1377*;    -   E129V+K177L+R179E+K220P+N224L+Q254S;    -   E129V+K177L+R179E+K220P+N224L+Q254S+M284T;    -   E129V+K177L+R179E+S242Q;    -   E129V+K177L+R179V+K220P+N224L+S242Q+Q254S;    -   K220P+N224L+S242Q+Q254S;    -   M284V;    -   V59A Q89R+E129V+K177L+R179E+Q254S+M284V.

In a preferred embodiment the alpha-amylase is selected from the groupof Bacillus stearothermophilus alpha-amylase variants:

-   -   I181*+G182*+N193F+E129V+K177L+R179E;    -   I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S    -   I181*+G182*+N193F+V59A Q89R+E129V+K177L+R179E+Q254S+M284V; and    -   I181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S        (using SEQ ID NO: 1 for numbering).

According to the invention the alpha-amylase variant has at least 80%,more preferably at least 85%, more preferably at least 90%, morepreferably at least 91%, more preferably at least 92%, even morepreferably at least 93%, most preferably at least 94%, and even mostpreferably at least 95%, such as even at least 96%, at least 97%, atleast 98%, at least 99%, but less than 100% identity to the mature partof the polypeptide of SEQ ID NO: 1 herein.

In another embodiment the alpha-amylase is a Bacillus licheniformisalpha-amylase, or a variant thereof. In an embodiment the Bacilluslicheniformis alpha-amylase is the one shown in SEQ ID NO: 21 herein.According to the invention the alpha-amylase has at least 80%, morepreferably at least 85%, more preferably at least 90%, more preferablyat least 91%, more preferably at least 92%, even more preferably atleast 93%, most preferably at least 94%, and even most preferably atleast 95%, such as even at least 96%, at least 97%, at least 98%, atleast 99% identity to the mature part of the polypeptide of SEQ ID NO:21 herein.

The alpha-amylase may according to the invention be present and/or addedin a concentration of 0.1-100 micro gram per gram DS, such as 0.5-50micro gram per gram DS, such as 1-25 micro gram per gram DS, such as1-10 micro gram per gram DS, such as 2-5 micro gram per gram DS.

In an embodiment from 1-10 micro gram Pyrococcus furiosus protease and1-10 micro gram Bacillus stearothermophilus alpha-amylase are presentand/or added in liquefaction.

Glucoamylase Present and/or Added in Liquefaction

In an embodiment a glucoamylase is present and/or added in liquefactionstep a) in a process of the invention (i.e., oil recovery process andfermentation product production process).

In a preferred embodiment the glucoamylase present and/or added inliquefaction has a heat stability at 85° C., pH 5.3, of at least 20%,such as at least 30%, preferably at least 35% determined as disclosed inExample 2 herein or Example 8 in WO 2011/127802

In an embodiment the glucoamylase has a relative activity pH optimum atpH 5.0 of at least 90%, preferably at least 95%, preferably at least 97%determined as disclosed in Example 2 herein or Example 8 in WO2011/127802.

In an embodiment the glucoamylase has a pH stability at pH 5.0 of atleast at least 80%, at least 85%, at least 90% determined as disclosedin Example 2 herein or Example 8 in WO 2011/127802.

In a preferred embodiment the glucoamylase present and/or added inliquefaction step a) is derived from a strain of the genus Penicillium,especially a strain of Penicillium oxalicum disclosed as SEQ ID NO: 2 inWO 2011/127802 or SEQ ID NOs: 9 or 14 herein.

In an embodiment the glucoamylase has at least 80%, more preferably atleast 85%, more preferably at least 90%, more preferably at least 91%,more preferably at least 92%, even more preferably at least 93%, mostpreferably at least 94%, and even most preferably at least 95%, such aseven at least 96%, at least 97%, at least 98%, at least 99% or 100%identity to the mature polypeptide shown in SEQ ID NO: 2 in WO2011/127802 or SEQ ID NOs: 9 or 14 herein.

In an embodiment the glucoamylase has a thermostability of at least 80°C., preferably at least 82° C., such as at least 84° C., such as atleast 86° C., such as at least 88° C. at pH 4.0 determined asDifferential Scanning Calorimitry (DSC) as described in Example 3 below.

In an embodiment the glucoamylase has a thermostability of at least 80°C., preferably at least 82° C., such as at least 84° C., such as atleast 86° C., such as at least 88° C., such as at least 90° C. at pH 4.8determined as Differential Scanning Calorimitry (DSC) as described inExample 3 below.

Examples of specifically contemplated glucoamylases can be found inExample 3 (Table 6) below.

In a preferred embodiment the glucoamylase is a variant of thePenicillium oxalicum glucoamylase shown in SEQ ID NO: 2 in WO2011/127802 or SEQ ID NO: 14 herein having a K79V substitution (usingthe mature sequence shown in SEQ ID NO: 14 for numbering), such as avariant disclosed in WO 2013/053801 (hereby incorporated by reference).

In an embodiment the Penicillium oxalicum glucoamylase has a K79Vsubstitution (using SEQ ID NO: 14 for numbering) and preferably furtherone of the following substitutions:

T65A; or

Q327F; or

E501V; or

Y504T; or

Y504*; or

T65A+Q327F; or

T65A+E501V; or

T65A+Y504T; or

T65A+Y504*; or

Q327F+E501V; or

Q327F+Y504T; or

Q327F+Y504*; or

E501V+Y504T; or

E501V+Y504*; or

T65A+Q327F+E501V; or

T65A+Q327F+Y504T; or

T65A+E501V+Y504T; or

Q327F+E501V+Y504T; or

T65A+Q327F+Y504*; or

T65A+E501V+Y504*; or

Q327F+E501V+Y504*; or

T65A+Q327F+E501V+Y504T; or

T65A+Q327F+E501V+Y504*;

E501V+Y504T; or

T65A+K161S; or

T65A+Q405T; or

T65A+Q327W; or

T65A+Q327F; or

T65A+Q327Y; or

P11F+T65A+Q327F; or

R1K+D3W+K5Q+G7V+N8S+T10K+P11S+T65A+Q327F; or

P2N+P4S+P11F+T65A+Q327F; or

P11F+D26C+K330+T65A+Q327F; or

P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; or

R1E+D3N+P4G+G6R+G7A+N8A+T10D+P11D+T65A+Q327F; or

P11F+T65A+Q327W; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or

P11F+T65A+Q327W+E501V+Y504T; or

T65A+Q327F+E501V+Y504T; or

T65A+S105P+Q327W; or

T65A+S105P+Q327F; or

T65A+Q327W+S364P; or

T65A+Q327F+S364P; or

T65A+S103N+Q327F; or

P2N+P4S+P11F+K34Y+T65A+Q327F; or

P2N+P4S+P11F+T65A+Q327F+D445N+V447S; or

P2N+P4S+P11F+T65A+I172V+Q327F; or

P2N+P4S+P11F+T65A+Q327F+N502*; or

P2N+P4S+P11F+T65A+Q327F+N502T+P563S+K571E; or

P2N+P4S+P11F+R31S+K33V+T65A+Q327F+N564D+K571S; or

P2N+P4S+P11F+T65A+Q327F+S377T; or

P2N+P4S+P11F+T65A+V325T+Q327W; or

P2N+P4S+P11F+T65A+Q327F+D445N+V447S+E501V+Y504T; or

P2N+P4S+P11F+T65A+I172V+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+S377T+E501V+Y504T; or

P2N+P4S+P11F+D26N+K34Y+T65A+Q327F; or

P2N+P4S+P11F+T65A+Q327F+I375A+E501V+Y504T; or

P2N+P4S+P11F+T65A+K218A+K221D+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+S103N+Q327F+E501V+Y504T; or

P2N+P4S+T10D+T65A+Q327F+E501V+Y504T; or

P2N+P4S+F12Y+T65A+Q327F+E501V+Y504T; or

K5A+P11F+T65A+Q327F+E501V+Y504T; or

P2N+P4S+T10E+E18N+T65A+Q327F+E501V+Y504T; or

P2N+T10E+E18N+T65A+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T568N; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+K524T+G526A; or

P2N+P4S+P11F+K34Y+T65A+Q327F+D445N+V447S+E501V+Y504T; or

P2N+P4S+P11F+R31S+K33V+T65A+Q327F+D445N+V447S+E501V+Y504T; or

P2N+P4S+P11F+D26N+K34Y+T65A+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+F80*+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+K112S+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T516P+K524T+G526A; or

P2N+P4S+P11F+T65A+Q327F+E501V+N502T+Y504*; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+S103N+Q327F+E501V+Y504T; or

K5A+P11F+T65A+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T516P+K524T+G526A; or

P2N+P4S+P11F+T65A+K79A+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+K79G+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+K791+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+K79L+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+K79S+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+L72V+Q327F+E501V+Y504T; or

S255N+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+E74N+V79K+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+G220N+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Y245N+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q253N+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+D279N+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+S359N+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+D370N+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+V460S+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+V460T+P468T+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+T463N+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+S465N+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+T477N+E501V+Y504T.

In a preferred embodiment the glucoamylase present and/or added inliquefaction is the Penicillium oxalicum glucoamylase having a K79Vsubstitution and preferably further one of the following substitutions:

-   -   P11F+T65A+Q327F;    -   P2N+P4S+P11F+T65A+Q327F (using SEQ ID NO: 14 for numbering).

In an embodiment the glucoamylase variant has at least 75% identitypreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 91%, more preferably at least92%, even more preferably at least 93%, most preferably at least 94%,and even most preferably at least 95%, such as even at least 96%, atleast 97%, at least 98%, at least 99%, but less than 100% identity tothe mature part of the polypeptide of SEQ ID NO: 14 herein.

The glucoamylase may be added in amounts from 0.1-100 micro grams EP/g,such as 0.5-50 micro grams EP/g, such as 1-25 micrograms EP/g, such as2-12 micrograms EP/g DS.

Glucoamylase Present and/or Added in Saccharification and/orFermentation

A glucoamylase is present and/or added in saccharification and/orfermentation, preferably simultaneous saccharification and fermentation(SSF), in a process of the invention (i.e., oil recovery process andfermentation product production process).

In an embodiment the glucoamylase present and/or added insaccharification and/or fermentation is of fungal origin, preferablyfrom a stain of Aspergillus, preferably A. niger, A. awamori, or A.oryzae; or a strain of Trichoderma, preferably T. reesei; or a strain ofTalaromyces, preferably T. emersonii.

In an embodiment the glucoamylase is derived from Talaromyces, such as astrain of Talaromyces emersonii, such as the one shown in SEQ ID NO: 19herein,

In an embodiment the glucoamylase is selected from the group consistingof:

(i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 19herein;

(ii) a glucoamylase comprising an amino acid sequence having at least60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to the mature polypeptide of SEQ ID NO: 19 herein.

In an embodiment the glucoamylase is derived from a strain of the genusPycnoporus, in particular a strain of Pycnoporus sanguineus described inWO 2011/066576 (SEQ ID NOs 2, 4 or 6), such as the one shown as SEQ IDNO: 4 in WO 2011/066576 or SEQ ID NO: 18 herein.

In an embodiment the glucoamylase is derived from a strain of the genusGloeophyllum, such as a strain of Gloeophyllum sepiarium or Gloeophyllumtrabeum, in particular a strain of Gloeophyllum as described in WO2011/068803 (SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16). In a preferredembodiment the glucoamylase is the Gloeophyllum sepiarium shown in SEQID NO: 2 in WO 2011/068803 or SEQ ID NO: 15 herein.

In a preferred embodiment the glucoamylase is derived from Gloephyllumserpiarium, such as the one shown in SEQ ID NO: 15 herein. In anembodiment the glucoamylase is selected from the group consisting of:

(i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 15herein;

(ii) a glucoamylase comprising an amino acid sequence having at least60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to the mature polypeptide of SEQ ID NO: 15 herein.

In another embodiment the glucoamylase is derived from Gloeophyllumtrabeum such as the one shown in SEQ ID NO: 17 herein. In an embodimentthe glucoamylase is selected from the group consisting of:

(i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 17herein;

(ii) a glucoamylase comprising an amino acid sequence having at least60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to the mature polypeptide of SEQ ID NO: 17 herein.

In an embodiment the glucoamylase is derived from a strain of the genusNigrofomes, in particular a strain of Nigrofomes sp. disclosed in WO2012/064351 (SEQ ID NO: 2) (all references hereby incorporated byreference).

Glucoamylases may in an embodiment be added to the saccharificationand/or fermentation in an amount of 0.0001-20 AGU/g DS, preferably0.001-10 AGU/g DS, especially between 0.01-5 AGU/g DS, such as 0.1-2AGU/g DS.

Commercially available compositions comprising glucoamylase include AMG200L; AMG 300 L; SAN™ SUPER, SAN™ EXTRA L, SPIRIZYME™ PLUS, SPIRIZYME™FUEL, SPIRIZYME™ B4U, SPIRIZYME™ ULTRA, SPIRIZYME™ EXCEL and AMG™ E(from Novozymes A/S); OPTIDEX™ 300, GC480, GC417 (from DuPont); AMIGASE™and AMIGASE™ PLUS (from DSM); G-ZYME™ G900, G-ZYME™ and G990 ZR (fromDuPont).

According to a preferred embodiment of the invention the glucoamylase ispresent and/or added in saccharification and/or fermentation incombination with an alpha-amylase. Examples of suitable alpha-amylaseare described below.

Alpha-Amylase Present and/or Added in Saccharification and/orFermentation

In an embodiment an alpha-amylase is present and/or added insaccharification and/or fermentation in a process of the invention. In apreferred embodiment the alpha-amylase is of fungal or bacterial origin.In a preferred embodiment the alpha-amylase is a fungal acid stablealpha-amylase. A fungal acid stable alpha-amylase is an alpha-amylasethat has activity in the pH range of 3.0 to 7.0 and preferably in the pHrange from 3.5 to 6.5, including activity at a pH of about 4.0, 4.5,5.0, 5.5, and 6.0.

In a preferred embodiment the alpha-amylase present and/or added insaccharification and/or fermentation is derived from a strain of thegenus Rhizomucor, preferably a strain the Rhizomucor pusillus, such asone shown in SEQ ID NO: 3 in WO 2013/006756, such as a Rhizomucorpusillus alpha-amylase hybrid having an Aspergillus niger linker andstarch-bonding domain, such as the one shown in SEQ ID NO: 16 herein, ora variant thereof.

In an embodiment the alpha-amylase present and/or added insaccharification and/or fermentation is selected from the groupconsisting of:

(i) an alpha-amylase comprising the mature polypeptide of SEQ ID NO: 16herein;

(ii) an alpha-amylase comprising an amino acid sequence having at least60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to the mature polypeptide of SEQ ID NO: 16 herein.

In a preferred embodiment the alpha-amylase is a variant of thealpha-amylase shown in SEQ ID NO: 13 having at least one of thefollowing substitutions or combinations of substitutions: D165M; Y141W;Y141R; K136F; K192R; P224A; P224R; S123H+Y141W; G20S+Y141W; A76G+Y141W;G128D+Y141W; G128D+D143N; P219C+Y141W; N142D+D143N; Y141W+K192R;Y141W+D143N; Y141W+N383R; Y141W+P219C+A265C; Y141W+N142D+D143N;Y141W+K192R V410A; G128D+Y141W+D143N; Y141W+D143N+P219C;Y141W+D143N+K192R; G128D+D143N+K192R; Y141W+D143N+K192R+P219C;G128D+Y141W+D143N+K192R; or G128D+Y141W+D143N+K192R+P219C (using SEQ IDNO: 16 for numbering).

In an embodiment the alpha-amylase is derived from a Rhizomucor pusilluswith an Aspergillus niger glucoamylase linker and starch-binding domain(SBD), preferably disclosed as SEQ ID NO: 13 herein, preferably havingone or more of the following substitutions: G128D, D143N, preferablyG128D+D143N (using SEQ ID NO: 13 for numbering).

In an embodiment the alpha-amylase variant present and/or added insaccharification and/or fermentation has at least 75% identitypreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 91%, more preferably at least92%, even more preferably at least 93%, most preferably at least 94%,and even most preferably at least 95%, such as even at least 96%, atleast 97%, at least 98%, at least 99%, but less than 100% identity tothe mature part of the polypeptide of SEQ ID NO: 16 herein.

In an embodiment the alpha-amylase is derived from a strain ofAspergillus, such as Aspergillus niger, such as the one shown as SEQ IDNO: 9 in U.S. Pat. No. 8,048,657; or Aspergillus kawachi, such as theone shown as SEQ ID NO: 5 in U.S. Pat. No. 8,048,657.

In an embodiment the alpha-amylase is derived from a strain ofTrichoderma reesei, such as the one shown in SEQ ID NO: 13 in U.S. Pat.No. 8,048,657.

In a preferred embodiment the ratio between glucoamylase andalpha-amylase present and/or added during saccharification and/orfermentation may preferably be in the range from 500:1 to 1:1, such asfrom 250:1 to 1:1, such as from 100:1 to 1:1, such as from 100:2 to100:50, such as from 100:3 to 100:70.

Pullulanase Present and/or Added in Liquefaction and/or Saccharificationand/or Fermentation.

A pullulanase may be present and/or added during liquefaction step a)and/or saccharification step b) or fermentation step c) or simultaneoussaccharification and fermentation.

Pullulanases (E.C. 3.2.1.41, pullulan 6-glucano-hydrolase), aredebranching enzymes characterized by their ability to hydrolyze thealpha-1,6-glycosidic bonds in, for example, amylopectin and pullulan.

Contemplated pullulanases according to the present invention include thepullulanases from Bacillus amyloderamificans disclosed in U.S. Pat. No.4,560,651 (hereby incorporated by reference), the pullulanase disclosedas SEQ ID NO: 2 in WO 01/151620 (hereby incorporated by reference), theBacillus deramificans disclosed as SEQ ID NO: 4 in WO 01/151620 (herebyincorporated by reference), and the pullulanase from Bacillusacidopullulyticus disclosed as SEQ ID NO: 6 in WO 01/151620 (herebyincorporated by reference) and also described in FEMS Mic. Let. (1994)115, 97-106.

Additional pullulanases contemplated according to the present inventionincluded the pullulanases from Pyrococcus woesei, specifically fromPyrococcus woesei DSM No. 3773 disclosed in WO92/02614.

In an embodiment the pullulanase is a family GH57 pullulanase, whereinthe pullulanase preferably includes an X47 domain as disclosed in WO2011/087836. More specifically the the pullulanase may be derived from astrain from the genus Thermococcus, including Thermococcus litoralis andThermococcus hydrothermalis or a hybrid thereof. In an embodiment thepullulanase is the truncated Thermococcus hydrothermalis pullulanase atsite X4 or a T. hydrothermalis/T. litoralis hybrid enzyme withtruncation site X4 disclosed in WO 2011/087836 or shown in SEQ ID NO: 12herein.

In another embodiment the pullulanase is one comprising an X46 domaindisclosed in WO 2011/076123 (Novozymes).

The pullulanase may according to the invention be added in an effectiveamount which include the preferred amount of about 0.0001-10 mg enzymeprotein per gram DS, preferably 0.0001-0.10 mg enzyme protein per gramDS, more preferably 0.0001-0.010 mg enzyme protein per gram DS.Pullulanase activity may be determined as NPUN. An Assay fordetermination of NPUN is described in the “Materials & Methods”-sectionbelow.

Suitable commercially available pullulanase products include PROMOZYMED, PROMOZYME™ D2 (Novozymes A/S, Denmark), OPTIMAX L-300 (Genencor Int.,USA), and AMANO 8 (Amano, Japan).

Further Aspects of Processes of the Invention

Prior to liquefaction step a), processes of the invention, includingprocesses of extracting/recovering oil and processes for producingfermentation products, may comprise the steps of:

-   -   i) reducing the particle size of the starch-containing material,        preferably by dry milling;    -   ii) forming a slurry comprising the starch-containing material        and water.

In an embodiment at least 50%, preferably at least 70%, more preferablyat least 80%, especially at least 90% of the starch-containing materialfit through a sieve with #6 screen.

In an embodiment the pH during liquefaction is between above 4.5-6.5,such as 4.5-5.0, such as around 4.8, or a pH between 5.0-6.2, such as5.0-6.0, such as between 5.0-5.5, such as around 5.2, such as around5.4, such as around 5.6, such as around 5.8.

In an embodiment the temperature during liquefaction is above theinitial gelatinization temperature, preferably in the range from 70-100°C., such as between 75-95° C., such as between 75-90° C., preferablybetween 80-90° C., especially around 85° C.

In an embodiment a jet-cooking step is carried out before liquefactionin step a). In an embodiment the jet-cooking is carried out at atemperature between 110-145° C., preferably 120-140° C., such as125-135° C., preferably around 130° C. for about 1-15 minutes,preferably for about 3-10 minutes, especially around about 5 minutes.

In a preferred embodiment saccharification and fermentation is carriedout sequentially or simultaneously.

In an embodiment saccharification is carried out at a temperature from20-75° C., preferably from 40-70° C., such as around 60° C., and at a pHbetween 4 and 5.

In an embodiment fermentation or simultaneous saccharification andfermentation (SSF) is carried out carried out at a temperature from 25°C. to 40° C., such as from 28° C. to 35° C., such as from 30° C. to 34°C., preferably around about 32° C. In an embodiment fermentation isongoing for 6 to 120 hours, in particular 24 to 96 hours.

In a preferred embodiment the fermentation product is recovered afterfermentation, such as by distillation.

In an embodiment the fermentation product is an alcohol, preferablyethanol, especially fuel ethanol, potable ethanol and/or industrialethanol.

In an embodiment the starch-containing starting material is wholegrains. In an embodiment the starch-containing material is selected fromthe group of corn, wheat, barley, rye, milo, sago, cassava, manioc,tapioca, sorghum, rice, and potatoes.

In an embodiment the fermenting organism is yeast, preferably a strainof Saccharomyces, especially a strain of Saccharomyces cerevisae.

In an embodiment the alpha-amylase is a bacterial or fungalalpha-amylase.

In an embodiment saccharification step b) and fermentation step c) arecarried out simultaneously or sequentially.

In an embodiment the temperature in step (a) is above the initialgelatinization temperature, such as at a temperature between 80-90° C.,such as around 85° C.

In an embodiment a process of the invention further comprises apre-saccharification step, before saccharification step b), carried outfor 40-90 minutes at a temperature between 30-65° C. In an embodimentsaccharification is carried out at a temperature from 20-75° C.,preferably from 40-70° C., such as around 60° C., and at a pH between 4and 5. In an embodiment fermentation step c) or simultaneoussaccharification and fermentation (SSF) (i.e., steps b) and c)) arecarried out carried out at a temperature from 25° C. to 40° C., such asfrom 28° C. to 35° C., such as from 30° C. to 34° C., preferably aroundabout 32° C. In an embodiment the fermentation step c) or simultaneoussaccharification and fermentation (SSF) (i.e., steps b) and c)) areongoing for 6 to 120 hours, in particular 24 to 96 hours.

In an embodiment separation in step e) is carried out by centrifugation,preferably a decanter centrifuge, filtration, preferably using a filterpress, a screw press, a plate-and-frame press, a gravity thickener ordecker.

In an embodiment the fermentation product is recovered by distillation.

Examples of Specific Process Embodiments of the Invention

Oil Recovery:

In a preferred embodiment the invention concerns processes of recoveringoil comprising the steps of:

a) liquefying starch-containing material at a temperature above theinitial gelatinization temperature using:

-   -   Bacillus stearothermophilus alpha-amylase comprising a double        deletion at positions I181+G182 using SEQ ID NO: 1 for        numbering;    -   more than 0.5 micro gram Pyrococcus furiosus protease per gram        dry solids (DS);    -   Penicillium oxalicum shown in SEQ ID NO: 14 comprising a K79V        substitution;

b) saccharifying using a glucoamylase;

c) fermenting using a fermenting organism.

d) recovering the fermentation product to form whole stillage;

e) separating the whole stillage into thin stillage and wet cake;

f) optionally concentrating the thin stillage into syrup;

wherein oil is recovered from the:

-   -   liquefied starch-containing material after step a); and/or    -   downstream from fermentation step c).

In a preferred embodiment the invention concerns processes of recoveringoil comprising the steps of:

a) liquefying starch-containing material at a temperature above theinitial gelatinization temperature using:

-   -   Bacillus stearothermophilus alpha-amylase comprising a double        deletion at positions:    -   I181+G182 and the following substitutions        N193F+V59A+Q89R+E129V+K177L+R179E+Q254S+M284V truncated to 491        amino acids (using SEQ ID NO: 1 for numbering).    -   more than 0.5 micro gram Pyrococcus furiosus protease per gram        dry solids (DS);    -   Penicillium oxalicum glucoamylase having the following        mutations: K79V+P2N+P4S+P11F+T65A+Q327F (using SEQ ID NO: 14 for        numbering);

b) saccharifying using a glucoamylase;

c) fermenting using a fermenting organism.

d) recovering the fermentation product to form whole stillage;

e) separating the whole stillage into thin stillage and wet cake;

f) optionally concentrating the thin stillage into syrup;

wherein oil is recovered from the:

-   -   liquefied starch-containing material after step a); and/or    -   downstream from fermentation step c).

In a preferred embodiment the ratio between alpha-amylase andglucoamylase in liquefaction is between 1:1 and 1:10, such as around 1:2(micro gram alpha-amylase per g DS:micro gram glucoamylase per gram DS).

In a preferred embodiment the ratio between alpha-amylase and proteasein liquefaction is in the range between 1:1 and 1:25, such between 1:1.2and as 1:10, such as around 1:1.4 (micro gram alpha-amylase per gramDS:micro gram protease per gram DS).

Producing Fermentation Products:

In a preferred embodiment the invention relates to processes forproducing fermentation products from starch-containing materialcomprising the steps of:

a) liquefying the starch-containing material at a temperature above theinitial gelatinization temperature using:

-   -   an alpha-amylase derived from Bacillus stearothermophilus;    -   more than 2 micro gram Pyrococcus furiosus protease per gram dry        solids (DS); and    -   optionally a Penicillium oxalicum glucoamylase;

b) saccharifying using a glucoamylase enzyme;

c) fermenting using a fermenting organism.

In a preferred embodiment the invention relates to processes forproducing fermentation products from starch-containing materialcomprising the steps of:

a) liquefying the starch-containing material at a temperature above theinitial gelatinization temperature using:

-   -   an alpha-amylase, preferably derived from Bacillus        stearothermophilus, having a T½ (min) at pH 4.5, 85° C., 0.12 mM        CaCl₂ of at least 10;    -   more than 2 micro gram Pyrococcus furiosus protease per gram dry        solids (DS); and    -   optionally a glucoamylase;

b) saccharifying using a glucoamylase enzyme;

c) fermenting using a fermenting organism.

In a preferred embodiment the invention relates to processes forproducing fermentation products from starch-containing materialcomprising the steps of:

a) liquefying the starch-containing material at a temperature above theinitial gelatinization temperature using:

-   -   an alpha-amylase, preferably derived from Bacillus        stearothermophilus, having a T½ (min) at pH 4.5, 85° C., 0.12 mM        CaCl₂) of at least 10;    -   more than 2 micro gram Pyrococcus furiosus protease per gram dry        solids (DS); and    -   a Penicillium oxalicum glucoamylasea;

b) saccharifying using a glucoamylase enzyme;

c) fermenting using a fermenting organism.

In a preferred embodiment the invention relates to processes forproducing fermentation products from starch-containing materialcomprising the steps of:

-   -   a) liquefying the starch-containing material at a temperature        above the initial gelatinization temperature using:        -   an alpha-amylase derived from Bacillus stearothermophilus            having a double deletion at positions I181+G182, and            optional substitution N193F; further one of the following            set of substitutions:            -   E129V+K177L+R179E;            -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;            -   V59A+Q89R+E129V+K177L+R179E+Q254S+M284V;            -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID                NO: 1 herein for numbering);        -   more than 2 micro gram Pyrococcus furiosus protease per gram            dry solids (DS);        -   a Penicillium oxalicum glucoamylase in SEQ ID NO: 14 having            substitutions selected from the group of:        -   K79V;        -   K79V+P11F+T65A+Q327F; or        -   K79V+P2N+P4S+P11F+T65A+Q327F; or        -   K79V+P11F+D26C+K33C+T65A+Q327F; or        -   K79V+P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; or        -   K79V+P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or        -   K79V+P11F+T65A+Q327W+E501V+Y504T (using SEQ ID NO: 14 for            numbering);    -   b) saccharifying using a glucoamylase enzyme;    -   c) fermenting using a fermenting organism.

In a preferred embodiment the invention relates to processes forproducing fermentation products from starch-containing materialcomprising the steps of:

-   -   a) liquefying the starch-containing material at a pH in the        range between from above 4.5-6.5 at a temperature between        80-90° C. using:        -   an alpha-amylase derived from Bacillus stearothermophilus            having a double deletion I181+G182 and optional substitution            N193F; and further one of the following set of            substitutions:            -   E129V+K177L+R179E;            -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;            -   V59A+Q89R+E129V+K177L+R179E+Q254S+M284V;            -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID                NO: 1 herein for numbering);        -   more than 2 micro gram, such as between 2-5 micro gram,            preferably around or more than 3 micro gram Pyrococcus            furiosus protease per gram DS dry solids (DS);        -   a Penicillium oxalicum glucoamylase in SEQ ID NO: 14 having            substitutions selected from the group of:        -   K79V;        -   K79V+P11F+T65A+Q327F; or        -   K79V+P2N+P4S+P11F+T65A+Q327F; or        -   K79V+P11F+D26C+K330+T65A+Q327F; or        -   K79V+P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; or        -   K79V+P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or        -   K79V+P11F+T65A+Q327W+E501V+Y504T (using SEQ ID NO: 14 for            numbering);    -   b) saccharifying using a glucoamylase enzyme;    -   c) fermenting using a fermenting organism.

In a preferred embodiment the invention relates to processes forproducing fermentation products from starch-containing materialcomprising the steps of:

-   -   a) liquefying the starch-containing material at a pH in the        range between from above 4.5-6.5 at a temperature between        80-90° C. using:        -   an alpha-amylase derived from Bacillus stearothermophilus            having a double deletion I181+G182 and substitution N193F;            and further one of the following set of substitutions:            -   E129V+K177L+R179E;            -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;            -   V59A+Q89R+E129V+K177L+R179E+Q254S+M284V;            -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID                NO: 1 herein for numbering);        -   more than 2 micro gram, such as between 2-5 micro gram,            preferably around or more than 3 micro gram Pyrococcus            furiosus protease per gram DS dry solids (DS)        -   a Penicillium oxalicum glucoamylase in SEQ ID NO: 14 having            substitutions selected from the group of:        -   K79V;        -   K79V+P11F+T65A+Q327F; or        -   K79V+P2N+P4S+P11F+T65A+Q327F; or        -   K79V+P11F+D26C+K33C+T65A+Q327F; or        -   K79V+P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; or        -   K79V+P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or        -   K79V+P11F+T65A+Q327W+E501V+Y504T (using SEQ ID NO: 14 for            numbering);    -   b) saccharifying using a Rhizomucor pusillus glucoamylase with        an Aspergillus niger glucoamylase linker and starch-binding        domain (SBD), preferably disclosed as SEQ ID NO: 13 herein,        preferably having one or more of the following substitutions:        G128D, D143N, preferably G128D+D143N (using SEQ ID NO: 13 for        numbering);    -   c) fermenting using a fermenting organism.

In an embodiment the ratio between alpha-amylase and glucoamylase inliquefaction is between 1:1 and 1:10, such as around 1:2 (micro gramalpha-amylase per g DS:micro gram glucoamylase per gram DS).

In an embodiment the ratio between alpha-amylase and protease inliquefaction is in the range between 1:1 and 1:25, such between 1:1.2and as 1:10, such as around 1:1.4 (micro gram alpha-amylase per gDS:micro gram protease per gram DS).

Fermentation Medium

The environment in which fermentation is carried out is often referredto as the “fermentation media” or “fermentation medium”. Thefermentation medium includes the fermentation substrate, that is, thecarbohydrate source that is metabolized by the fermenting organism.According to the invention the fermentation medium may comprisenutrients and growth stimulator(s) for the fermenting organism(s).Nutrient and growth stimulators are widely used in the art offermentation and include nitrogen sources, such as ammonia; urea,vitamins and minerals, or combinations thereof.

Fermenting Organisms

The term “fermenting organism” refers to any organism, includingbacterial and fungal organisms, especially yeast, suitable for use in afermentation process and capable of producing the desired fermentationproduct. Especially suitable fermenting organisms are able to ferment,i.e., convert, sugars, such as glucose or maltose, directly orindirectly into the desired fermentation product, such as ethanol.Examples of fermenting organisms include fungal organisms, such asyeast. Preferred yeast includes strains of Saccharomyces spp., inparticular, Saccharomyces cerevisiae.

Suitable concentrations of the viable fermenting organism duringfermentation, such as SSF, are well known in the art or can easily bedetermined by the skilled person in the art. In one embodiment thefermenting organism, such as ethanol fermenting yeast, (e.g.,Saccharomyces cerevisiae) is added to the fermentation medium so thatthe viable fermenting organism, such as yeast, count per mL offermentation medium is in the range from 10⁵ to 10¹², preferably from10⁷ to 10¹⁰, especially about 5×10⁷.

Examples of commercially available yeast includes, e.g., RED START™ andETHANOL RED™ yeast (available from Fermentis/Lesaffre, USA), FALI(available from Fleischmann's Yeast, USA), SUPERSTART and THERMOSACC™fresh yeast (available from Ethanol Technology, WI, USA), BIOFERM AFTand XR (available from NABC—North American Bioproducts Corporation, GA,USA), GERT STRAND (available from Gert Strand AB, Sweden), and FERMIOL(available from DSM Specialties).

Starch-Containing Materials

Any suitable starch-containing material may be used according to thepresent invention. The starting material is generally selected based onthe desired fermentation product. Examples of starch-containingmaterials, suitable for use in a process of the invention, include wholegrains, corn, wheat, barley, rye, milo, sago, cassava, tapioca, sorghum,rice, peas, beans, or sweet potatoes, or mixtures thereof or starchesderived therefrom, or cereals. Contemplated are also waxy and non-waxytypes of corn and barley. In a preferred embodiment thestarch-containing material, used for ethanol production according to theinvention, is corn or wheat.

Fermentation Products

The term “fermentation product” means a product produced by a processincluding a fermentation step using a fermenting organism. Fermentationproducts contemplated according to the invention include alcohols (e.g.,ethanol, methanol, butanol; polyols such as glycerol, sorbitol andinositol); organic acids (e.g., citric acid, acetic acid, itaconic acid,lactic acid, succinic acid, gluconic acid); ketones (e.g., acetone);amino acids (e.g., glutamic acid); gases (e.g., H₂ and CO₂); antibiotics(e.g., penicillin and tetracycline); enzymes; vitamins (e.g.,riboflavin, B₁₂, beta-carotene); and hormones. In a preferred embodimentthe fermentation product is ethanol, e.g., fuel ethanol; drinkingethanol, i.e., potable neutral spirits; or industrial ethanol orproducts used in the consumable alcohol industry (e.g., beer and wine),dairy industry (e.g., fermented dairy products), leather industry andtobacco industry. Preferred beer types comprise ales, stouts, porters,lagers, bitters, malt liquors, happoushu, high-alcohol beer, low-alcoholbeer, low-calorie beer or light beer. Preferably processes of theinvention are used for producing an alcohol, such as ethanol. Thefermentation product, such as ethanol, obtained according to theinvention, may be used as fuel, which is typically blended withgasoline. However, in the case of ethanol it may also be used as potableethanol.

Recovery of Fermentation Products

Subsequent to fermentation, or SSF, the fermentation product may beseparated from the fermentation medium. The slurry may be distilled toextract the desired fermentation product (e.g., ethanol). Alternativelythe desired fermentation product may be extracted from the fermentationmedium by micro or membrane filtration techniques. The fermentationproduct may also be recovered by stripping or other method well known inthe art.

Recovery of Oil

According to the invention oil is recovered during and/or afterliquefying, from the whole stillage, from the thin stillage or from thesyrup. Oil may be recovered by extraction. In one embodiment oil isrecovered by hexane extraction. Other oil recovery technologieswell-known in the art may also be used.

An Enzyme Composition of the Invention

An enzyme composition of the invention comprises an alpha-amylase and aPyrococcus furiosus protease suitable for use in a liquefaction step ina process of the invention.

An enzyme composition of the invention comprises:

-   -   i) Bacillus sp. alpha-amylase, or a variant thereof;    -   ii) Pyrococcus furiosus protease;

wherein the ratio between alpha-amylase and protease is in the rangefrom 1:1 and 1:25 (micro gram alpha-amylase:micro gram protease).

In a preferred embodiment the ratio between alpha-amylase and proteaseis in the range between 1:1.2 and 1:10, such as around 1:1.4 (micro gramalpha-amylase:micro gram protease).

In a preferred embodiment the enzyme composition of the inventioncomprises a glucoamylase and the ratio between alpha-amylase andglucoamylase in liquefaction is between 1:1 and 1:10, such as around 1:2(micro gram alpha-amylase:micro gram glucoamylase).

In an embodiment the alpha-amylase in the enzyme composition of theinvention is a bacterial or fungal alpha-amylase.

In an embodiment the alpha-amylase is from the genus Bacillus, such as astrain of Bacillus stearothermophilus, in particular a variant of aBacillus stearothermophilus alpha-amylase, such as the one shown in SEQID NO: 3 in WO 99/019467 or SEQ ID NO: 1 herein.

In an embodiment the Bacillus stearothermophilus alpha-amylase orvariant thereof is truncated, preferably to have around 491 amino acids,such as from 480-495 amino acids.

In an embodiment the Bacillus stearothermophilus alpha-amylase has adouble deletion, preferably at positions I181+G182 and optionally aN193F substitution, or double deletion of R179 and G180 (using SEQ IDNO: 1 for numbering).

In an embodiment the Bacillus stearothermophilus alpha-amylase has asubstitution at position S242, preferably S242Q substitution.

In an embodiment the Bacillus stearothermophilus alpha-amylase has asubstitution at position E188, preferably E188P substitution.

In an embodiment the alpha-amylase has a T½ (min) at pH 4.5, 85° C.,0.12 mM CaCl₂) of at least 10, such as at least 15, such as at least 20,such as at least 25, such as at least 30, such as at least 40, such asat least 50, such as at least 60, such as between 10-70, such as between15-70, such as between 20-70, such as between 25-70, such as between30-70, such as between 40-70, such as between 50-70, such as between60-70.

In an embodiment the alpha-amylase is selected from the group ofBacillus stearothermphilus alpha-amylase variants with the followingmutations:

-   -   I181*+G182*+N193F+E129V+K177L+R179E;    -   I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;    -   I181*+G182*+N193F+V59A Q89R+E129V+K177L+R179E+Q254S+M284V; and    -   I181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S        (using SEQ ID NO: 1 herein for numbering).

In an embodiment the alpha-amylase variant has at least 75% identitypreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 91%, more preferably at least92%, even more preferably at least 93%, most preferably at least 94%,and even most preferably at least 95%, such as even at least 96%, atleast 97%, at least 98%, at least 99%, but less than 100% identity tothe mature part of the polypeptide of SEQ ID NO: 1 herein.

In another embodiment the alpha-amylase is a Bacillus licheniformisalpha-amylase, or a variant thereof.

In an embodiment the Bacillus licheniformis alpha-amylase is the oneshown in SEQ ID NO: 21 herein.

In an embodiment the alpha-amylase has at least 80%, more preferably atleast 85%, more preferably at least 90%, more preferably at least 91%,more preferably at least 92%, even more preferably at least 93%, mostpreferably at least 94%, and even most preferably at least 95%, such aseven at least 96%, at least 97%, at least 98%, at least 99% identity tothe mature part of the polypeptide of SEQ ID NO: 21 herein.

In an embodiment the enzyme composition comprises a Bacilluslicheniformis alpha-amylase and a Pyrococcus furiosus protease.

In an embodiment the enzyme composition further comprises aglucoamylase.

In an embodiment the Pyrococcus furiosus is the one shown in SEQ ID NO:13 herein.

In an embodiment the Pyrococcus furiosus protease is one having at least80%, such as at least 85%, such as at least 90%, such as at least 95%,such as at least 96%, such as at least 97%, such as at least 98%, suchas at least 99% identity to SEQ ID NO: 13 herein.

In an embodiment the enzyme composition further comprises a glucoamylaseshown in SEQ ID NO: 14, or a variant thereof.

In an embodiment the glucoamylase has a heat stability at 85° C., pH5.3, of at least 20%, such as at least 30%, preferably at least 35%determined as disclosed in Example 2 herein or Example 8 in WO2011/127802.

In an embodiment the glucoamylase has a relative activity pH optimum atpH 5.0 of at least 90%, preferably at least 95%, preferably at least 97%determined as disclosed in Example 2 herein or Example 8 in WO2011/127802.

In an embodiment the glucoamylase has a pH stability at pH 5.0 of atleast at least 80%, at least 85%, at least 90% determined as disclosedin Example 2 herein or Example 8 in WO 2011/127802.

In an embodiment the glucoamylase is derived from a strain of the genusPenicillium, especially a strain of Penicillium oxalicum disclosed asSEQ ID NO: 2 in WO 2011/127802 or SEQ ID NOs: 9 or 14 herein.

In an embodiment the glucoamylase has at least 80%, more preferably atleast 85%, more preferably at least 90%, more preferably at least 91%,more preferably at least 92%, even more preferably at least 93%, mostpreferably at least 94%, and even most preferably at least 95%, such aseven at least 96%, at least 97%, at least 98%, at least 99% or 100%identity to the mature polypeptide shown in SEQ ID NO: 2 in WO2011/127802 or SEQ ID NOs: 9 or 14 herein.

In an embodiment the glucoamylase has a thermostability of at least 80°C., preferably at least 82° C., such as at least 84° C., such as atleast 86° C., such as at least 88° C. at pH 4.0 determined asDifferential Scanning Calorimitry (DSC) as described in Example 3 below.

In an embodiment the glucoamylase has a thermostability of at least 80°C., preferably at least 82° C., such as at least 84° C., such as atleast 86° C., such as at least 88° C., such as at least 90° C. at pH 4.8determined as Differential Scanning Calorimitry (DSC) as described inExample 3 below.

Examples of specifically contemplated glucoamylases can be found inExample 3 (Table 6) below.

In an embodiment the glucoamylase is a variant of the Penicilliumoxalicum glucoamylase disclosed as SEQ ID NO: 2 in WO 2011/127802 or SEQID NO: 14 herein having a K79V substitution (using the mature sequenceshown in SEQ ID NO: 14 for numbering) such as a variant disclosed in WO2013/053801.

In an embodiment the Penicillium oxalicum glucoamylase has a K79Vsubstitution (using SEQ ID NO: 14 for numbering) and preferably furtherone of the following substitutions:

T65A; or

Q327F; or

E501V; or

Y504T; or

Y504*; or

T65A+Q327F; or

T65A+E501V; or

T65A+Y504T; or

T65A+Y504*; or

Q327F+E501V; or

Q327F+Y504T; or

Q327F+Y504*; or

E501V+Y504T; or

E501V+Y504*; or

T65A+Q327F+E501V; or

T65A+Q327F+Y504T; or

T65A+E501V+Y504T; or

Q327F+E501V+Y504T; or

T65A+Q327F+Y504*; or

T65A+E501V+Y504*; or

Q327F+E501V+Y504*; or

T65A+Q327F+E501V+Y504T; or

T65A+Q327F+E501V+Y504*;

E501V+Y504T; or

T65A+K161S; or

T65A+Q405T; or

T65A+Q327W; or

T65A+Q327F; or

T65A+Q327Y; or

P11F+T65A+Q327F; or

R1K+D3W+K5Q+G7V+N8S+T10K+P11S+T65A+Q327F; or

P2N+P4S+P11F+T65A+Q327F; or

P11F+D26C+K33C+T65A+Q327F; or

P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; or

R1E+D3N+P4G+G6R+G7A+N8A+T10D+P11D+T65A+Q327F; or

P11F+T65A+Q327W; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or

P11F+T65A+Q327W+E501V+Y504T; or

T65A+Q327F+E501V+Y504T; or

T65A+S105P+Q327W; or

T65A+S105P+Q327F; or

T65A+Q327W+S364P; or

T65A+Q327F+S364P; or

T65A+S103N+Q327F; or

P2N+P4S+P11F+K34Y+T65A+Q327F; or

P2N+P4S+P11F+T65A+Q327F+D445N+V447S; or

P2N+P4S+P11F+T65A+I172V+Q327F; or

P2N+P4S+P11F+T65A+Q327F+N502*; or

P2N+P4S+P11F+T65A+Q327F+N502T+P563S+K571E; or

P2N+P4S+P11F+R31S+K33V+T65A+Q327F+N564D+K571S; or

P2N+P4S+P11F+T65A+Q327F+S377T; or

P2N+P4S+P11F+T65A+V325T+Q327W; or

P2N+P4S+P11F+T65A+Q327F+D445N+V447S+E501V+Y504T; or

P2N+P4S+P11F+T65A+I172V+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+S377T+E501V+Y504T; or

P2N+P4S+P11F+D26N+K34Y+T65A+Q327F; or

P2N+P4S+P11F+T65A+Q327F+I375A+E501V+Y504T; or

P2N+P4S+P11F+T65A+K218A+K221D+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+S103N+Q327F+E501V+Y504T; or

P2N+P4S+T10D+T65A+Q327F+E501V+Y504T; or

P2N+P4S+F12Y+T65A+Q327F+E501V+Y504T; or

K5A+P11F+T65A+Q327F+E501V+Y504T; or

P2N+P4S+T10E+E18N+T65A+Q327F+E501V+Y504T; or

P2N+T10E+E18N+T65A+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T568N; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+K524T+G526A; or

P2N+P4S+P11F+K34Y+T65A+Q327F+D445N+V447S+E501V+Y504T; or

P2N+P4S+P11F+R31S+K33V+T65A+Q327F+D445N+V447S+E501V+Y504T; or

P2N+P4S+P11F+D26N+K34Y+T65A+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+F80*+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+K112S+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T516P+K524T+G526A; or

P2N+P4S+P11F+T65A+Q327F+E501V+N502T+Y504*; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+S103N+Q327F+E501V+Y504T; or

K5A+P11F+T65A+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T516P+K524T+G526A; or

P2N+P4S+P11F+T65A+K79A+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+K79G+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+K791+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+K79L+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+K79S+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+L72V+Q327F+E501V+Y504T; or

S255N+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+E74N+V79K+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+G220N+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Y245N+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q253N+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+D279N+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+S359N+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+D370N+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+V460S+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+V460T+P468T+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+T463N+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+S465N+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+T477N+E501V+Y504T.

In an embodiment the glucoamylase is the Penicillium oxalicumglucoamylase having a K79V substitution (using SEQ ID NO: 14 fornumbering) and further one of the following substitutions:

-   -   P11F+T65A+Q327F    -   P2N+P4S+P11F+T65A+Q327F (using SEQ ID NO: 14 for numbering).

In an embodiment the glucoamylase variant has at least 75% identitypreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 91%, more preferably at least92%, even more preferably at least 93%, most preferably at least 94%,and even most preferably at least 95%, such as even at least 96%, atleast 97%, at least 98%, at least 99%, but less than 100% identity tothe mature part of the polypeptide of SEQ ID NO: 14 herein.

In an embodiment the composition further comprising a pullulanase.

In an embodiment the pullulanase is a family GH57 pullulanase, whereinthe pullulanase preferably includes an X47 domain as disclosed in WO2011/087836.

In an embodiment the pullulanase is derived from a strain from the genusThermococcus, including Thermococcus litoralis and Thermococcushydrothermalis or a hybrid thereof.

In an embodiment the pullulanase is the truncated Thermococcushydrothermalis pullulanase at site X4 or a T. hydrothermalis/T.litoralis hybrid enzyme with truncation site X4 disclosed in WO2011/087836 or shown in SEQ ID NO: 12 herein.

In an embodiment the enzyme composition comprises

-   -   Bacillus stearothermophilus alpha-amylase, or a variant thereof;    -   Pyrococcus furiosus protease; and    -   Penicillium oxalicum glucoamylase,

wherein the ratio between alpha-amylase and protease is in the rangefrom 1:1 and 1:25 (micro gram alpha-amylase:micro gram protease).

In an embodiment the enzyme composition of the invention comprises:

-   -   an alpha-amylase, preferably derived from Bacillus        stearothermophilus, having a T½ (min) at pH 4.5, 85° C., 0.12 mM        CaCl₂ of at least 10;    -   Pyrococcus furiosus protease; and    -   Penicillium oxalicum glucoamylase,

wherein the ratio between alpha-amylase and protease is in the rangefrom 1:1 and 1:25 (micro gram alpha-amylase:micro gram protease).

In an embodiment the enzyme composition comprises:

-   -   an alpha-amylase derived from Bacillus stearothermophilus having        a double deletion I181+G182 and substitution N193F; and further        one of the following set of substitutions:        -   E129V+K177L+R179E;        -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;        -   V59A+Q89R+E129V+K177L+R179E+Q254S+M284V;        -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID NO:            1 herein for numbering);    -   Pyrococcus furiosus protease; and    -   Penicillium oxalicum glucoamylase in SEQ ID NO: 14 having        substitutions selected from the group of:    -   K79V;    -   K79V+P11F+T65A+Q327F; or    -   K79V+P2N+P4S+P11F+T65A+Q327F; or    -   K79V+P11F+D26C+K33C+T65A+Q327F; or    -   K79V+P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; or    -   K79V+P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or    -   K79V+P11F+T65A+Q327W+E501V+Y504T (using SEQ ID NO: 14 for        numbering),

wherein the ratio between alpha-amylase and protease is in the rangefrom 1:1 and 1:25 (micro gram alpha-amylase:micro gram protease).

In an embodiment the ratio between alpha-amylase and protease is in therange between 1:1.2 and 1:10, such as around 1:1.4 (micro gramalpha-amylase:micro gram protease).

In an embodiment the ratio between alpha-amylase and glucoamylase isbetween 1:1 and 1:10, such as around 1:2 (micro gram alpha-amylase:microgram glucoamylase).

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Materials & Methods

Materials:

Alpha-Amylase A (AAA): Bacillus stearothermophilus alpha-amylase withthe mutations I181*+G182*+N193F truncated to 491 amino acids (SEQ ID NO:1)

Alpha-Amylase 1407 (AA1407): Bacillus stearothermophilus alpha-amylasewith the mutationsI181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254Struncated to 491 amino acids (SEQ ID NO: 1)

Alpha-Amylase 369 (AA369): Bacillus stearothermophilus alpha-amylasewith the mutations:I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+Q254S+M284V truncated to491 amino acids (SEQ ID NO: 1).

Protease Pfu: Protease derived from Pyrococcus furiosus shown in SEQ IDNO: 13 herein.

Glucoamylase Po: Mature part of the Penicillium oxalicum glucoamylasedisclosed as SEQ ID NO: 2 in WO 2011/127802 and shown in SEQ ID NO: 14herein.

Glucoamylase Po PE001: Variant of Penicillium oxalicum glucoamylasehaving the following mutation: K79V (using SEQ ID NO: 14 for numbering).

Glucoamylase Po 498 (GA498): Variant of Penicillium oxalicumglucoamylase having the following mutations:K79V+P2N+P4S+P11F+T65A+Q327F (using SEQ ID NO: 14 for numbering).

Glucoamylase A: Blend comprising Talaromyces emersonii glucoamylasedisclosed as SEQ ID NO: 34 in WO99/28448, Trametes cingulataglucoamylase disclosed as SEQ ID NO: 2 in WO 06/69289, and Rhizomucorpusillus alpha-amylase with Aspergillus niger glucoamylase linker andstarch binding domain (SBD) disclosed in SEQ ID NO: 16 herein having thefollowing substitutions G128D+D143N (activity ratio in AGU:AGU:FAU-F isabout 20:5:1).

Glucoamylase U: Blend comprising Talaromyces emersonii glucoamylasedisclosed as SEQ ID NO: 34 in WO99/28448, Trametes cingulataglucoamylase disclosed as SEQ ID NO: 2 in WO 06/69289 and Rhizomucorpusillus alpha-amylase with Aspergillus niger glucoamylase linker andstarch binding domain (SBD) disclosed disclosed in SEQ ID NO: 16 herein(activity ratio in AGU:AGU:FAU-F is about 65:15:1).

Protease X: Metallo protease derived from Thermoascus aurantiacus CGMCCNo. 0670 disclosed as amino acids 1-177 in SEQ ID NO: 3 herein and aminoacids 1-177 in SEQ ID NO: 2 in WO 2003/048353

Yeast:

ETHANOL RED™ from Fermentis, USA

Methods

Identity:

The relatedness between two amino acid sequences or between twonucleotide sequences is described by the parameter “identity”.

For purposes of the present invention the degree of identity between twoamino acid sequences, as well as the degree of identity between twonucleotide sequences, may be determined by the program “align” which isa Needleman-Wunsch alignment (i.e. a global alignment). The program isused for alignment of polypeptide, as well as nucleotide sequences. Thedefault scoring matrix BLOSUM50 is used for polypeptide alignments, andthe default identity matrix is used for nucleotide alignments. Thepenalty for the first residue of a gap is −12 for polypeptides and −16for nucleotides. The penalties for further residues of a gap are −2 forpolypeptides, and −4 for nucleotides.

“Align” is part of the FASTA package version v20u6 (see W. R. Pearsonand D. J. Lipman (1988), “Improved Tools for Biological SequenceAnalysis”, PNAS 85:2444-2448, and W. R. Pearson (1990) “Rapid andSensitive Sequence Comparison with FASTP and FASTA,” Methods inEnzymology 183:63-98). FASTA protein alignments use the Smith-Watermanalgorithm with no limitation on gap size (see “Smith-Watermanalgorithm”, T. F. Smith and M. S. Waterman (1981) J. Mol. Biol.147:195-197).

Protease Assays

AZCL-Casein Assay

A solution of 0.2% of the blue substrate AZCL-casein is suspended inBorax/NaH₂PO₄ buffer pH9 while stirring. The solution is distributedwhile stirring to microtiter plate (100 microL to each well), 30 microLenzyme sample is added and the plates are incubated in an EppendorfThermomixer for 30 minutes at 45° C. and 600 rpm. Denatured enzymesample (100° C. boiling for 20 min) is used as a blank. After incubationthe reaction is stopped by transferring the microtiter plate onto iceand the colored solution is separated from the solid by centrifugationat 3000 rpm for 5 minutes at 4° C. 60 microL of supernatant istransferred to a microtiter plate and the absorbance at 595 nm ismeasured using a BioRad Microplate Reader.

pNA-Assay

50 microL protease-containing sample is added to a microtiter plate andthe assay is started by adding 100 microL 1 mM pNA substrate (5 mgdissolved in 100 microL DMSO and further diluted to 10 mL withBorax/NaH₂PO₄ buffer pH 9.0). The increase in OD₄₀₅ at room temperatureis monitored as a measure of the protease activity.

Glucoamylase Activity (AGU)

Glucoamylase activity may be measured in Glucoamylase Units (AGU).

The Novo Glucoamylase Unit (AGU) is defined as the amount of enzyme,which hydrolyzes 1 micromole maltose per minute under the standardconditions 37° C., pH 4.3, substrate: maltose 23.2 mM, buffer: acetate0.1 M, reaction time 5 minutes.

An autoanalyzer system may be used. Mutarotase is added to the glucosedehydrogenase reagent so that any alpha-D-glucose present is turned intobeta-D-glucose. Glucose dehydrogenase reacts specifically withbeta-D-glucose in the reaction mentioned above, forming NADH which isdetermined using a photometer at 340 nm as a measure of the originalglucose concentration.

AMG incubation: Substrate: maltose 23.2 mM Buffer: acetate 0.1M pH: 4.30± 0.05 Incubation temperature: 37° C. ± 1 Reaction time: 5 minutesEnzyme working range: 0.5-4.0 AGU/mL

Color reaction: GlucDH: 430 U/L Mutarotase: 9 U/L NAD: 0.21 mM Buffer:phosphate 0.12M; 0.15M NaCl pH: 7.60 ± 0.05 Incubation temperature: 37°C. ± 1 Reaction time: 5 minutes Wavelength: 340 nm

A folder (EB-SM-0131.02/01) describing this analytical method in moredetail is available on request from Novozymes A/S, Denmark, which folderis hereby included by reference.

Acid Alpha-Amylase Activity

-   -   When used according to the present invention the activity of an        acid alpha-amylase may be measured in AFAU (Acid Fungal        Alpha-amylase Units) or FAU-F.

Acid Alpha-Amylase Activity (AFAU)

-   -   Acid alpha-amylase activity may be measured in AFAU (Acid Fungal        Alpha-amylase Units), which are determined relative to an enzyme        standard. 1 AFAU is defined as the amount of enzyme which        degrades 5.260 mg starch dry matter per hour under the below        mentioned standard conditions.    -   Acid alpha-amylase, an endo-alpha-amylase        (1,4-alpha-D-glucan-glucanohydrolase, E.C. 3.2.1.1) hydrolyzes        alpha-1,4-glucosidic bonds in the inner regions of the starch        molecule to form dextrins and oligosaccharides with different        chain lengths. The intensity of color formed with iodine is        directly proportional to the concentration of starch. Amylase        activity is determined using reverse colorimetry as a reduction        in the concentration of starch under the specified analytical        conditions.

Standard conditions/reaction conditions: Substrate: Soluble starch,approx. 0.17 g/L Buffer: Citrate, approx. 0.03M Iodine (I2): 0.03 g/LCaCl₂: 1.85 mM pH: 2.50 ± 0.05 Incubation temperature: 40° C. Reactiontime: 23 seconds Wavelength: 590 nm Enzyme concentration: 0.025 AFAU/mLEnzyme working range: 0.01-0.04 AFAU/mL

-   -   A folder EB-SM-0259.02/01 describing this analytical method in        more detail is available upon request to Novozymes A/S, Denmark,        which folder is hereby included by reference.

Determination of FAU-F

-   -   FAU-F Fungal Alpha-Amylase Units (Fungamyl) is measured relative        to an enzyme standard of a declared strength.

Reaction conditions Temperature 37° C. pH 7.15 Wavelength 405 nmReaction time 5 min Measuring time 2 min

-   -   A folder (EB-SM-0216.02) describing this standard method in more        detail is available on request from Novozymes A/S, Denmark,        which folder is hereby included by reference.

Alpha-Amylase Activity (KNU)

The alpha-amylase activity may be determined using potato starch assubstrate. This method is based on the break-down of modified potatostarch by the enzyme, and the reaction is followed by mixing samples ofthe starch/enzyme solution with an iodine solution. Initially, ablackish-blue color is formed, but during the break-down of the starchthe blue color gets weaker and gradually turns into a reddish-brown,which is compared to a colored glass standard.

One Kilo Novo alpha amylase Unit (KNU) is defined as the amount ofenzyme which, under standard conditions (i.e., at 37° C.+/−0.05; 0.0003M Ca²⁺; and pH 5.6) dextrinizes 5260 mg starch dry substance MerckAmylum solubile.

A folder EB-SM-0009.02/01 describing this analytical method in moredetail is available upon request to Novozymes A/S, Denmark, which folderis hereby included by reference.

Determination of Pullulanase Activity (NPUN)

Endo-pullulanase activity in NPUN is measured relative to a Novozymespullulanase standard. One pullulanase unit (NPUN) is defined as theamount of enzyme that releases 1 micro mol glucose per minute under thestandard conditions (0.7% red pullulan (Megazyme), pH 5, 40° C., 20minutes). The activity is measured in NPUN/ml using red pullulan.

1 mL diluted sample or standard is incubated at 40° C. for 2 minutes.0.5 mL 2% red pullulan, 0.5 M KCl, 50 mM citric acid, pH 5 are added andmixed. The tubes are incubated at 40° C. for 20 minutes and stopped byadding 2.5 ml 80% ethanol. The tubes are left standing at roomtemperature for 10-60 minutes followed by centrifugation 10 minutes at4000 rpm. OD of the supernatants is then measured at 510 nm and theactivity calculated using a standard curve.

The present invention is described in further detail in the followingexamples which are offered to illustrate the present invention, but notin any way intended to limit the scope of the invention as claimed. Allreferences cited herein are specifically incorporated by reference forthat which is described therein.

EXAMPLES Example 1

Stability of Alpha-Amylase Variants

The stability of a reference alpha-amylase (Bacillus stearothermophilusalpha-amylase with the mutations I181*+G182*+N193F truncated to 491amino acids (SEQ ID NO: 1 numbering)) and alpha-amylase variants thereofwas determined by incubating the reference alpha-amylase and variants atpH 4.5 and 5.5 and temperatures of 75° C. and 85° C. with 0.12 mM CaCl₂followed by residual activity determination using the EnzChek® substrate(EnzChek® Ultra Amylase assay kit, E33651, Molecular Probes).

Purified enzyme samples were diluted to working concentrations of 0.5and 1 or 5 and 10 ppm (micrograms/ml) in enzyme dilution buffer (10 mMacetate, 0.01% Triton X100, 0.12 mM CaCl₂, pH 5.0). Twenty microlitersenzyme sample was transferred to 48-well PCR MTP and 180 microlitersstability buffer (150 mM acetate, 150 mM MES, 0.01% Triton X100, 0.12 mMCaCl₂, pH 4.5 or 5.5) was added to each well and mixed. The assay wasperformed using two concentrations of enzyme in duplicates. Beforeincubation at 75° C. or 85° C., 20 microliters was withdrawn and storedon ice as control samples. Incubation was performed in a PCR machine at75° C. and 85° C. After incubation samples were diluted to 15 ng/mL inresidual activity buffer (100 mM Acetate, 0.01% Triton X100, 0.12 mMCaCl₂, pH 5.5) and 25 microliters diluted enzyme was transferred toblack 384-MTP. Residual activity was determined using the EnzCheksubstrate by adding 25 microliters substrate solution (100micrograms/ml) to each well. Fluorescence was determined every minutefor 15 minutes using excitation filter at 485-P nm and emission filterat 555 nm (fluorescence reader is Polarstar, BMG). The residual activitywas normalized to control samples for each setup.

Assuming logarithmic decay half life time (T½ (min)) was calculatedusing the equation: T½ (min)=T(min)*LN(0.5)/LN(% RA/100), where T isassay incubation time in minutes, and % RA is % residual activitydetermined in assay.

Using this assay setup the half life time was determined for thereference alpha-amylase and variant thereof as shown in Table 1.

TABLE 1 T½ T½ T½ (min) (min) (min) (pH 5.5, (pH 4.5, (pH 4.5, 85° C.,75° C., 85° C., 0.12 0.12 mM 0.12 mM mM Mutations CaCl₂) CaCl₂) CaCl₂)Reference Alpha-Amylase A 21 4 111 Reference Alpha-Amylase A with 32 6301 the substitution V59A Reference Alpha-Amylase A with 28 5 230 thesubstitution V59E Reference Alpha-Amylase A with 28 5 210 thesubstitution V59I Reference Alpha-Amylase A with 30 6 250 thesubstitution V59Q Reference Alpha-Amylase A with 149 22 ND thesubstitutions V59A + Q89R + G112D + E129V + K177L + R179E + K220P +N224L + Q254S Reference Alpha-Amylase A with >180 28 ND thesubstitutions V59A + Q89R + E129V + K177L + R179E + H208Y + K220P +N224L + Q254S Reference Alpha-Amylase A with 112 16 ND the substitutionsV59A + Q89R + E129V + K177L + R179E + K220P + N224L + Q254S + D269E +D281N Reference Alpha-Amylase A with 168 21 ND the substitutions V59A +Q89R + E129V + K177L + R179E + K220P + N224L + Q254S + I270L ReferenceAlpha-Amylase A with >180 24 ND the substitutions V59A + Q89R + E129V +K177L + R179E + K220P + N224L + Q254S + H274K Reference Alpha-Amylase Awith 91 15 ND the substitutions V59A + Q89R + E129V + K177L + R179E +K220P + N224L + Q254S + Y276F Reference Alpha-Amylase A with 141 41 NDthe substitutions V59A + E129V + R157Y + K177L + R179E + K220P + N224L +S242Q + Q254S Reference Alpha-Amylase A with >180 62 ND thesubstitutions V59A + E129V + K177L + R179E + H208Y + K220P + N224L +S242Q + Q254S Reference Alpha-Amylase A with >180 49 >480 thesubstitutions V59A + E129V + K177L + R179E + K220P + N224L + S242Q +Q254S Reference Alpha-Amylase A with >180 53 ND the substitutions V59A +E129V + K177L + R179E + K220P + N224L + S242Q + Q254S + H274K ReferenceAlpha-Amylase A with >180 57 ND the substitutions V59A + E129V + K177L +R179E + K220P + N224L + S242Q + Q254S + Y276F Reference Alpha-Amylase Awith >180 37 ND the substitutions V59A + E129V + K177L + R179E + K220P +N224L + S242Q + Q254S + D281N Reference Alpha-Amylase A with >180 51 NDthe substitutions V59A + E129V + K177L + R179E + K220P + N224L + S242Q +Q254S + M284T Reference Alpha-Amylase A with >180 45 ND thesubstitutions V59A + E129V + K177L + R179E + K220P + N224L + S242Q +Q254S + G416V Reference Alpha-Amylase A with 143 21 >480 thesubstitutions V59A + E129V + K177L + R179E + K220P + N224L + Q254SReference Alpha-Amylase A with >180 22 ND the substitutions V59A +E129V + K177L + R179E + K220P + N224L + Q254S + M284T ReferenceAlpha-Amylase A with >180 38 ND the substitutions A91L + M96I + E129V +K177L + R179E + K220P + N224L + S242Q + Q254S Reference Alpha-Amylase Awith 57 11 402 the substitutions E129V + K177L + R179E ReferenceAlpha-Amylase A with 174 44 >480 the substitutions E129V + K177L +R179E + K220P + N224L + S242Q + Q254S Reference Alpha-Amylase Awith >180 49 >480 the substitutions E129V + K177L + R179E + K220P +N224L + S242Q + Q254S + Y276F + L427M Reference Alpha-Amylase Awith >180 49 >480 the substitutions E129V + K177L + R179E + K220P +N224L + S242Q + Q254S + M284T Reference Alpha-Amylase A with 177 36 >480the substitutions E129V + K177L + R179E + K220P + N224L + S242Q +Q254S + N376* + I377* Reference Alpha-Amylase A with 94 13 >480 thesubstitutions E129V + K177L + R179E + K220P + N224L + Q254S ReferenceAlpha-Amylase A with 129 24 >480 the substitutions E129V + K177L +R179E + K220P + N224L + Q254S + M284T Reference Alpha-Amylase A with 14830 >480 the substitutions E129V + K177L + R179E + S242Q ReferenceAlpha-Amylase A with 78 9 >480 the substitutions E129V + K177L + R179VReference Alpha-Amylase A with 178 31 >480 the substitutions E129V +K177L + R179V + K220P + N224L + S242Q + Q254S Reference Alpha-Amylase Awith 66 17 >480 the substitutions K220P + N224L + S242Q + Q254SReference Alpha-Amylase A with 30 6 159 the substitutions K220P +N224L + Q254S Reference Alpha-Amylase A with 35 7 278 the substitutionM284T Reference Alpha-Amylase A with 59 13 ND the substitutions M284V NDnot determined

The results demonstrate that the alpha-amylase variants have asignificantly greater half-life and stability than the referencealpha-amylase.

Example 2

Characterization of Penicillium oxalicum Glucoamylase

The Penicillium oxalicum glucoamylase is disclosed in SEQ ID NO: 9herein.

Substrate.

Substrate: 1% soluble starch (Sigma S-9765) in deionized water

Reaction buffer: 0.1M Acetate buffer at pH 5.3

Glucose concentration determination kit: Wako glucose assay kit(LabAssay glucose, WAKO, Cat #298-65701).

Reaction Condition.

20 microL soluble starch and 50 microL acetate buffer at pH 5.3 weremixed. 30 microL enzyme solution (50 micro g enzyme protein/ml) wasadded to a final volume of 100 microL followed by incubation at 37° C.for 15 min.

The glucose concentration was determined by Wako kits.

All the work carried out in parallel.

Temperature Optimum.

To assess the temperature optimum of the Penicillium oxalicumglucoamylase the “Reaction condition”-assay described above wasperformed at 20, 30, 40, 50, 60, 70, 80, 85, 90 and 95° C. The resultsare shown in Table 2.

TABLE 2 Temperature optimum Temperature (° C.) 20 30 40 50 60 70 80 8590 95 Relative activity 63.6 71.7 86.4 99.4 94.6 100.0 92.9 92.5 82.782.8 (%)

From the results it can be seen that the optimal temperature forPenicillium oxalicum glucoamylase at the given conditions is between 50°C. and 70° C. and the glucoamylase maintains more than 80% activity at95° C.

Heat Stability.

To assess the heat stability of the Penicillium oxalicum glucoamylasethe Reaction condition assay was modified in that the the enzymesolution and acetate buffer was preincubated for 15 min at 20, 30, 40,50, 60, 70, 75, 80, 85, 90 and 95° C. Following the incubation 20 microLof starch was added to the solution and the assay was performed asdescribed above.

The results are shown in Table 3.

TABLE 3 Heat stability Temperature (° C.) 20 30 40 50 60 70 80 85 90 95Relative 91.0 92.9 88.1 100.0 96.9 86.0 34.8 36.0 34.2 34.8 activity (%)

From the results it can be seen that Penicillium oxalicum glucoamylaseis stable up to 70° C. after preincubation for 15 min in that itmaintains more than 80% activity.

pH Optimum.

To assess the pH optimum of the Penicillium oxalicum glucoamylase theReaction condition assay described above was performed at pH 2.0, 3.0,3.5, 4.0, 4.5, 5.0, 6.0 7.0, 8.0, 9.0, 10.0 and 11.0. Instead of usingthe acetate buffer described in the Reaction condition assay thefollowing buffer was used 100 mM Succinic acid, HEPES, CHES, CAPSO, 1 mMCaCl₂, 150 mM KCl, 0.01% Triton X-100, pH adjusted to 2.0, 3.0, 3.5,4.0, 4.5, 5.0, 6.0 7.0, 8.0, 9.0, 10.0 or 11.0 with HCl or NaOH.

The results are shown in Table 4.

TABLE 4 pH optimum pH 2.0 3.0 3.5 4.0 4.5 5.0 6.0 7.0 8.0 9.0 10.0 11.0Relative 71.4 78.6 77.0 91.2 84.2 100.0 55.5 66.7 30.9 17.8 15.9 16.1activity (%)

From the results it can be seen that Penicillium oxalicum glucoamylaseat the given conditions has the highest activity at pH 5.0. ThePenicillium oxalicum glucoamylase is active in a broad pH range in theit maintains more than 50% activity from pH 2 to 7.

pH Stability.

To assess the heat stability of the Penicillium oxalicum glucoamylasethe Reaction condition assay was modified in that the enzyme solution(50 micro g/mL) was preincubated for 20 hours in buffers with pH 2.0,3.0, 3.5, 4.0, 4.5, 5.0, 6.0 7.0, 8.0, 9.0, 10.0 and 11.0 using thebuffers described under pH optimum. After preincubation, 20 microLsoluble starch to a final volume of 100 microL was added to the solutionand the assay was performed as described above.

The results are shown in Table 5.

TABLE 5 pH stability pH 2.0 3.0 3.5 4.0 4.5 5.0 6.0 7.0 8.0 9.0 10.011.0 Relative 17.4 98.0 98.0 103.2 100.0 93.4 71.2 90.7 58.7 17.4 17.017.2 activity (%)

From the results it can be seen that Penicillium oxalicum glucoamylase,is stable from pH 3 to pH 7 after preincubation for 20 hours and itdecreases its activity at pH 8.

Example 3

Penicillium oxalicum Glucoamylase Variants (PoAMG)—ThermostabilityAnalysis by Differential Scanning Calorimitry (DSC)

Site specific Penicillium oxalicum glucoamylase (PoAMG) variants havingsubstitutions and/or deletions at specific positions were constructedbasically as described in Example 3 and purified as described in Example4 in WO2013/053801 (hereby incorporated by reference).

The thermostability of the purified Glucoamylase Po PE001 (SEQ ID NO: 14with K79V) derived variants were determined at pH 4.0 or 4.8 (50 mMSodium Acetate) by Differential Scanning calorimetry (DSC) using aVP-Capillary Differential Scanning calorimeter (MicroCal Inc.,Piscataway, N.J., USA). The thermal denaturation temperature, Td (° C.),was taken as the top of the denaturation peak (major endothermic peak)in thermograms (Cp vs. T) obtained after heating enzyme solutions inselected buffers (50 mM Sodium Acetate, pH 4.0 or 4.8) at a constantprogrammed heating rate of 200 K/hr.

Sample- and reference-solutions (approximately 0.3 ml) were loaded intothe calorimeter (reference: buffer without enzyme) from storageconditions at 10° C. and thermally pre-equilibrated for 10 minutes at20° C. prior to DSC scan from 20° C. to 110° C. Denaturationtemperatures were determined with an accuracy of approximately +/−1° C.

The isolated variants and the DSC data are disclosed in Table 6 below.

TABLE 6 Mutations DSC Td (° C.) @ DSC Td (° C.) @ Po-AMG name (+K79V) pH4.0 pH 4.8 Glucoamylase 82.1 83.4 Po PE001 SEQ ID NO: 14 with K79V PE167E501V Y504T 82.1 PE481 T65A K161S 84.1 86.0 PE487 T65A Q405T 83.2 PE490T65A Q327W 87.3 PE491 T65A Q327F 87.7 PE492 T65A Q327Y 87.3 PE493 P11FT65A Q327F 87.8 88.5 PE497 R1K D3W K5Q G7V N8S T10K P11S 87.8 88.0 T65AQ327F PE498 P2N P4S P11F T65A Q327F 88.3 88.4 PE003 P11F D26C K33C T65AQ327F 83.3 84.0 PE009 P2N P4S P11F T65A Q327W E501V 88.8 Y504T PE002 R1ED3N P4G G6R G7A N8A T10D 87.5 88.2 P11D T65A Q327F PE005 P11F T65A Q327W87.4 88.0 PE008 P2N P4S P11F T65A Q327F E501V 89.4 90.2 Y504T PE010 P11FT65A Q327W E501V Y504T 89.7 PE507 T65A Q327F E501V Y504T 89.3 PE513 T65AS105P Q327W 87.0 PE514 T65A S105P Q327F 87.4 PE515 T65A Q327W S364P 87.8PE516 T65A Q327F S364P 88.0 PE517 T65A S103N Q327F 88.9 PE022 P2N P4SP11F K34Y T65A Q327F 89.7 PE023 P2N P4S P11F T65A Q327F D445N 89.9 V447SPE032 P2N P4S P11F T65A I172V Q327F 88.7 PE049 P2N P4S P11F T65A Q327FN502* 88.4 PE055 P2N P4S P11F T65A Q327F N502T 88.0 P563S K571E PE057P2N P4S P11F R31S K33V T65A 89.5 Q327F N564D K571S PE058 P2N P4S P11FT65A Q327F S377T 88.6 PE064 P2N P4S P11F T65A V325T Q327W 88.0 PE068 P2NP4S P11F T65A Q327F D445N 90.2 V447S E501V Y504T PE069 P2N P4S P11F T65AI172V Q327F 90.2 E501V Y504T PE073 P2N P4S P11F T65A Q327F S377T 90.1E501V Y504T PE074 P2N P4S P11F D26N K34Y T65A 89.1 Q327F PE076 P2N P4SP11F T65A Q327F I375A 90.2 E501V Y504T PE079 P2N P4S P11F T65A K218AK221D 90.9 Q327F E501V Y504T PE085 P2N P4S P11F T65A S103N Q327F 91.3E501V Y504T PE086 P2N P4S T10D T65A Q327F E501V 90.4 Y504T PE088 P2N P4SF12Y T65A Q327F E501V 90.4 Y504T PE097 K5A P11F T65A Q327F E501V 90.0Y504T PE101 P2N P4S T10E E18N T65A Q327F 89.9 E501V Y504T PE102 P2N T10EE18N T65A Q327F E501V 89.8 Y504T PE084 P2N P4S P11F T65A Q327F E501V90.5 Y504T T568N PE108 P2N P4S P11F T65A Q327F E501V 88.6 Y504T K524TG526A PE126 P2N P4S P11F K34Y T65A Q327F 91.8 D445N V447S E501V Y504TPE129 P2N P4S P11F R31S K33V T65A 91.7 Q327F D445N V447S E501V Y504TPE087 P2N P4S P11F D26N K34Y T65A 89.8 Q327F E501V Y504T PE091 P2N P4SP11F T65A F80* Q327F 89.9 E501V Y504T PE100 P2N P4S P11F T65A K112SQ327F 89.8 E501V Y504T PE107 P2N P4S P11F T65A Q327F E501V 90.3 Y504TT516P K524T G526A PE110 P2N P4S P11F T65A Q327F E501V 90.6 N502T Y504*

Example 4

Use of High Dosage of Protease Pfu for Oil Extraction and Ethanol

Liquefaction:

Nine slurries of whole ground corn, backset and tap water were preparedto a total weight of 150 g targeting 32.50% Dry Solids (DS); backset wasblended at 30% weight of backset per weight of slurry. Slurry pH was 5.0and no further adjustments were made before applying the followingtreatments:

-   -   3 mashes were controls, meaning that they only received        Alpha-Amylase 369 (AA369) during liquefaction and will be the        baseline. AA369 was applied at a fixed dose of 2.1 μg/gDS in all        cases when applied.    -   2 mashes were treated with AA369 and 1.5 μg/gDS Protease Pfu.    -   2 mashes were treated with AA369 and 3 μg/gDS Protease Pfu.    -   2 mashes were treated with AA369 and 5 μg/gDS Protease Pfu.

Water and enzymes were added to each canister, and then each canisterwas sealed and mixed well prior to loading into the Labomat. All sampleswere incubated in the Labomat set to the following conditions: 5°C./min. Ramp, 15 minute Ramp to 80° C., hold for 1 min, Ramp to 85° C.at 1° C./min and holding for 103 min., 40 rpm for 30 seconds to the leftand 30 seconds to the right. Once liquefaction was complete, allcanisters were cooled in an ice bath for approximately 20 minutes beforeproceeding to fermentation.

Simultaneous Saccharification and Fermentation (SSF):

Penicillin was added to each mash to a final concentration of 3 ppm andadjusted to pH 5.0 with either 40% sulfuric acid or 45% potassiumhydroxide as needed. Next, a portion of this mash was transferred totest tubes and represents “urea-free” fermentations, or ones which areconsidered to be nitrogen limited. Once the “urea-free” mashes wereprocessed, the remaining mashes were dosed with urea up to a finalconcentration of 200 ppm and transferred to test tubes for fermentation.All test tubes were drilled with a 1/64″ bit to allow CO₂ release.Furthermore, equivalent solids were maintained across all treatmentsthrough the addition of water as required to ensure that the urea versusurea-free mashes contained equal solids. Fermentation was initiatedthrough the addition of Glucoamylase A (0.60 AGU/gDS), water andrehydrated yeast. Yeast rehydration took place by mixing 5.5 g ofFermentis' ETHANOL RED™ into 100 mL of 32° C. tap water for at least 15minutes and dosing 100 μl per test tube.

Distillation:

A Büchi Multivapor evaporation system was used for all distillations.The unit distilled 12 samples at a time. The parameters used are shownin Table 7. Tubes were weighed after distillation and weight lost duringdistillation was replaced with DI water. Tubes were weighed again afterwater addition. Three separate distillations were performed for thisexperiment which included a control each run.

TABLE 7 Distillation parameters for corn oil assay. Time 80 minTemperature 75° C. Vacuum 200-153 mBar (40 min) 153-148 mBar (40 min)RPM 8

Oil Extraction:

Hexane was added to each sample at a dose of 0.125 mL hexane/1 gstarting material. Each tube was covered in Dura-seal to prevent sampleleakage, and mixed thoroughly. Tubes were centrifuged at 3,000×g for 10minutes in an Avanti JE Series centrifuge with JS-5.3 rotor. Aftercentrifugation, the oil/hexane layer (supernatant) was removed using apositive displacement pipette, transferred to a pre-weighed 5 mLflip-top tube, and reweighed. The density of the sample was measuredusing a Rudolph Research Analytical density meter. The density of thesupernatant was then calculated using the standard curve equation tofind the % oil in the supernatant. From this value the total % oil inthe starting material was derived.

HPLC Analysis:

HPLC analysis used an Agilent 1100/1200 combined with a Bio-Rad HPX-87HIon Exclusion column (300 mm×7.8 mm) and a Bio-Rad Cation H guardcartridge. The mobile phase was 0.005 M sulfuric acid and processedsamples at a flow rate of 0.6 ml/min, with column and RI detectortemperatures of 65 and 55° C., respectively. Fermentation sampling tookplace after 54 hours by sacrificing 3 tubes per treatment. Each tube wasprocessed by deactivation with 50 μL of 40% v/v H₂SO₄, vortexing,centrifuging at 1460×g for 10 minutes, and filtering through a 0.45 μmWhatman PP filter. Samples were stored at 4° C. prior to and during HPLCanalysis. The method quantified analytes using calibration standards forDP4+, DP3, DP2, glucose, fructose, acetic acid, lactic acid, glyceroland ethanol (% w/v). A four point calibration including the origin isused for quantification.

Results (corn oil extraction): Terminology used in the example: For cornoil extraction, there are three separate controls (Control A, B and C)because each distillation processed a control for each run. However,each control was processed the same during liquefaction andfermentation: no protease during fermentation with Protease X additionto fermentation. Extraction of corn oil showed that the lowest dose ofProtease Pfu added to liquefaction matched the dose of Protease X(0.025% w/w) to fermentation (FIG. 1). Moreover, increasing Protease Pfuto 3 and 5 μg/gDS surpassed the oil recovered with Protease X alone, andthere was no additional benefit from combining both Protease Pfu andProtease X at this dose, suggesting little to no synergy between thesetwo proteases (FIGS. 2 & 3).

Ethanol: Performance of Various Proteases on Limited Dosage of ExogenousNitrogen Addition

The performance of various proteases using limiting dosage of exogenousnitrogen was also tested. It is investigates how the substrates producedfrom these proteases affect fermentation rate, carbohydrate consumptionand glycerol formation.

Treating fermentation with Protease X and operating without supplementalnitrogen from urea did not lead to dryness, whereas delivered dryness bythe 54 hour mark at all dosages (FIG. 4). Furthermore, increasingProtease Pfu to 3 or 5 μg/gDS from 1.5 μg/gDS led to a significantincrease in final ethanol concentration (% w/v) (FIGS. 4 & 5). Thesevarious protease treatments also demonstrate an effect on fermentationrate as the 24 hour ethanol concentrations are highest with the highesttreatment of Protease Pfu.

Similar trends were observed when incorporating 200 ppm urea intofermentation, but Protease Pfu activity during liquefaction remainedsuperior relative to Protease X by delivering the lowest residualglucose and yielding the highest final ethanol. Here, the lowest dose ofProtease Pfu (1.5 μg/gDS) outperformed Protease X by supporting a fasterfermentation through the first 24 hours, combined with statisticallyhigher final ethanol concentrations (% w/v) (FIGS. 6 & 7). In general,more Protease Pfu led to more ethanol production by the 54 hour mark,though the two highest doses were statistically equivalent in thisexample. All fermentations reached a state of dryness by the 54 hourmark. Higher doses of Protease Pfu also reduced the formation ofglycerol during fermentation, and this shift in metabolism is part ofthe reason why the increase in ethanol is being observed (FIG. 8—% w/v).

Conclusions

Oil Extraction:

-   -   1.5 μg/gDS of Protease Pfu action during a conventional corn        based liquefaction (85° C., pH 5.0, 2 hours) working in        combination with 2.1 μg EP/gDS Alpha-Amylase 369 matched the        increase in oil extracted from fermentation treatment with        Protease X (5 μg/gDS).    -   An even higher Protease Pfu dose of 3 μg/gDS led to        approximately 5% more oil versus Protease X alone and similar        results were seen with 5 μg/gDS dose as well.    -   There was no apparent synergy in oil recovery when combining        Protease Pfu with Protease X, and there seems to be no        improvement in running protease during liquefaction versus        fermentation.

Ethanol Yield:

Protease Pfu showed superior performance over Protease X

-   -   No Urea:        -   24 hour data showed Protease Pfu (1.5 μg/gDS) delivered a            much more efficient fermentation than Protease X by having            lower residual glucose and higher ethanol concentrations,            where increasing to 3 or 5 μg/gDS led to even more ethanol            production.        -   54 hour data showed Protease Pfu (1.5 μg/gDS) outperformed            Protease X by delivering low residual glucose and much            higher ethanol concentrations. Fermentations reached dryness            with all dosages of Protease Pfu, whereas Protease X            finished with just over 1% w/v.        -   Glycerol concentrations were 10% lower than Protease X with            Protease Pfu (5 μg/gDS).    -   200 ppm Urea:        -   24 hour data showed that Protease Pfu (1.5 μg/gDS)            outperformed Protease X by having lower residual glucose and            higher ethanol concentrations with 200 ppm urea. In general,            more Protease Pfu led to more ethanol by this time point,            though the two highest doses (3 and 5 μg/gDS) were very            similar.        -   54 hour HPLC showed that Protease Pfu (1.5 μg/gDS)            outperformed Protease X by delivering the lowest residual            glucose while yielding the highest final ethanol. All            fermentations reached dryness. Even the lowest dose of            Protease Pfu (1.5 μg/gDS) yielded 1% more ethanol than            Protease X. While 3 and 5 μg/gDS Protease Pfu were            statistically equivalent in final ethanol, they were both            higher than the lowest Protease Pfu dose.        -   Glycerol concentrations were approximately 9% lower than            Protease X with Protease Pfu (5 μg/gDS). Protease Pfu            delivered the best final ethanol concentrations while also            delivering the lowest final glycerol concentrations.

Example 5

Use of High Protease Pfu Dose in Liquefaction in Ethanol ProductionProcess

Liquefaction:

Thirteen slurries of whole ground corn and tap water were prepared to atotal weight of 125 g targeting 32.50% Dry Solids (DS); backset wasblended at 30% weight of backset per weight of slurry. Initial slurry pHwas approximately 6.0 and was adjusted to 5.0 with either 45% w/vpotassium hydroxide or 40% v/v sulfuric acid. A fixed dose ofAlpha-Amylase 1407 (1.73 μg EP/gDS) was applied to all slurries and wascombined with Protease Pfu as follows to evaluate the effect of highprotease treatment during liquefaction:

-   -   Control: Alpha-amylase Only    -   Alpha-amylase 1407+0.0355 μg/gDS Protease Pfu    -   Alpha-amylase 1407+0.25 μg/gDS Protease Pfu    -   Alpha-amylase 1047+0.5 μg/gDS Protease Pfu    -   Alpha-amylase 1407+1 μg/gDS Protease Pfu    -   Alpha-amylase 1407+10 μg/gDS Protease Pfu    -   Alpha-amylase 1407+50 μg/gDS Protease Pfu

Water and enzymes were added to each canister, and then each canisterwas sealed and mixed well prior to loading into the Labomat. All sampleswere incubated in the Labomat set to the following conditions: 5°C./min. Ramp, 15 minute Ramp to 80° C., hold for 1 min, Ramp to 85° C.at 1° C./min and holding for 103 min., 40 rpm for 30 seconds to the leftand 30 seconds to the right. Once liquefaction was complete, allcanisters were cooled in an ice bath for approximately 20 minutes beforeproceeding to fermentation.

Simultaneous Saccharification and Fermentation (SSF):

Penicillin was added to each mash to a final concentration of 3 ppm andpH was adjusted to 5.0. Next, portions of this mash were transferred totest tubes to represent “urea-free” fermentations and are considerednitrogen limited. Once the “urea-free” mashes were processed, theremaining mashes were dosed with urea up to a final concentration of 800ppm and also transferred to test tubes. All test tubes were drilled witha 1/64″ bit to allow CO₂ release. Furthermore, equivalent solids weremaintained across all treatments through the addition of water asrequired to ensure that the urea versus urea-free mashes contained equalsolids. Fermentation was initiated through the addition of GlucoamylaseU (0.50 AGU/gDS), water and rehydrated yeast. Yeast rehydration tookplace by mixing 5.5 g of ETHANOL RED™ into 100 mL of 32° C. tap waterfor at least 15 minutes and dosing 100 μl per test tube.

HPLC Analysis:

HPLC analysis used an Agilent 1100/1200 combined with a Bio-Rad HPX-87HIon Exclusion column (300 mm×7.8 mm) and a Bio-Rad Cation H guardcartridge. The mobile phase was 0.005 M sulfuric acid and processedsamples at a flow rate of 0.6 ml/min, with column and RI detectortemperatures of 65 and 55° C., respectively. Fermentation sampling tookplace after 54 hours by sacrificing 3 tubes per treatment. Each tube wasprocessed by deactivation with 50 μL of 40% v/v H₂SO₄, vortexing,centrifuging at 1460×g for 10 minutes, and filtering through a 0.45 μmWhatman PP filter. Samples were stored at 4° C. prior to and during HPLCanalysis. The method quantified analytes using calibration standards forDP4+, DP3, DP2, glucose, fructose, acetic acid, lactic acid, glyceroland ethanol (% w/v). A four point calibration including the origin isused for quantification.

Conclusions:

Comparison of 54 hour ethanol concentrations showed that more than 1μg/gDS Protease Pfu was required to support fermentation to drynessunder nitrogen limited conditions (FIGS. 9 & 10). Moreover, residualglucose was 0.4% w/v for the 1 μg/gDS dose, whereas no residual glucosewas observed at Protease Pfu dosages of 10 or 50 μg/gDS. The mostsignificant reduction in glycerol was observed at the two highestdosages of Protease Pfu, 10 and 50 μg/gDS. These results suggest that asmuch as 10 to 50 μg/gDS Protease Pfu may be required during liquefactionto achieve optimal performance as it relates to liquefaction andfermentation.

Summary Paragraphs

The present invention is defined in the claims and accompanyingdescription. For convenience, other aspects of the present invention arepresented herein by way of numbered paragraphs:

1. A process of recovering oil from a fermentation product productionprocess comprising the steps of:

-   -   a) liquefying starch-containing material at a temperature above        the initial gelatinization temperature using:        -   an alpha-amylase;        -   more than 0.5 micro gram Pyrococcus furiosus protease per            gram dry solids (DS);    -   b) saccharifying using a glucoamylase;    -   c) fermenting using a fermenting organism.    -   d) recovering the fermentation product to form whole stillage;    -   e) separating the whole stillage into thin stillage and wet        cake;    -   f) optionally concentrating the thin stillage into syrup;

wherein oil is recovered from the:

-   -   liquefied starch-containing material after step a); and/or    -   downstream from fermentation step c).

2. The process of paragraph 1, wherein oil is recovered during and/orafter liquefying the starch-containing material.

3. The process of paragraph 1, wherein oil is recovered from the wholestillage.

4. The process of any of paragraphs 1-3, wherein oil is recovered fromthe thin stillage.

5. The process of any of paragraphs 1-4, wherein oil is recovered fromthe syrup.

6. The process of any of paragraph 1-5, wherein 0.5-100 micro gramPyrococcus furiosus protease per gram DS, such as 1-50 micro gramPyrococcus furiosus protease per gram DS, such as 1-10 micro gramPyrococcus furiosus protease per gram DS, such as 1.5-5 micro gramPyrococcus furiosus protease per gram DS, such as around or more than1.5 micro gram Pyrococcus furiosus protease per gram DS are presentand/or added in liquefaction step a).

7. The process of any of paragraphs 1-6 wherein 2-100 micro gramPyrococcus furiosus protease per gram DS, such as 2.5-50 micro gramPyrococcus furiosus protease per gram DS, such as 2.5-10 micro gramPyrococcus furiosus protease per gram DS, such as 2.5-5 micro gramPyrococcus furiosus protease gram DS, especially around 3 micro gramPyrococcus furiosus protease per gram DS are present and/or added inliquefaction step a).

8. The process of any of paragraphs 1-7, wherein the Pyrococcus furiosusprotease is the mature sequence shown in SEQ ID NO: 13 herein.

9. The process of any of paragraphs 1-8, wherein the Pyrococcus furiosusprotease is one having at least 80%, such as at least 85%, such as atleast 90%, such as at least 95%, such as at least 96%, such as at least97%, such as at least 98%, such as at least 99% identity to SEQ ID NO:13 herein.

10. The process of any of paragraph 1-9, wherein no nitrogen-compound ispresent and/or added in steps a)-c), such as during saccharificationstep b), fermentation step c), or simultaneous saccharification andfermentation (SSF).

11. The process of any of paragraph 1-10, wherein 10-1,000 ppm, such as50-800 ppm, such as 100-600 ppm, such as 200-500 ppm nitrogen-compound,preferably urea, is present and/or added in steps a)-c), such as insaccharification step b) or fermentation step c) or in simultaneoussaccharification and fermentation (SSF).

12. The process of any of paragraphs 1-11, wherein the alpha-amylase isfrom the genus Bacillus, such as a strain of Bacillusstearothermophilus, such as the sequence shown in SEQ ID NO: 1.

13. The process of any of paragraphs 1-12, wherein the alpha-amylase aBacillus stearothermophilus alpha-amylase shown in SEQ ID NO: 1 herein,such as one having at least 80%, such as at least 85%, such as at least90%, such as at least 95%, such as at least 96%, such as at least 97%,such as at least 98%, such as at least 99% identity to SEQ ID NO: 1herein.

14. The process of paragraph 11, wherein the Bacillus stearothermophilusalpha-amylase or variant thereof is truncated, preferably to have around491 amino acids, such as from 480-495 amino acids.

15. The process of any of paragraphs 12-14, wherein the Bacillusstearothermophilus alpha-amylase has a double deletion at positionsI181+G182, and optionally a N193F substitution (using SEQ ID NO: 1 fornumbering),

16. The process of any of paragraphs 12-14, wherein the Bacillusstearothermophilus alpha-amylase has a double deletion at positionsR179+G180 and optionally a N193F substitution (using SEQ ID NO: 1 fornumbering).

17. The process of any of paragraphs 12-16, wherein the Bacillusstearothermophilus alpha-amylase has a substitution at position S242,preferably S242Q substitution.

18. The process of any of paragraphs 12-17, wherein the Bacillusstearothermophilus alpha-amylase has a substitution at position E188,preferably E188P substitution.

19. The process of any of paragraphs 1-18, wherein the alpha-amylase hasa T½ (min) at pH 4.5, 85° C., 0.12 mM CaCl₂) of at least 10.

20. The process of any of paragraphs 1-19, wherein the alpha-amylase hasa T½ (min) at pH 4.5, 85° C., 0.12 mM CaCl₂) of at least 15, such as atleast 20, such as at least 25, such as at least 30, such as at least 40,such as at least 50, such as at least 60, such as between 10-70, such asbetween 15-70, such as between 20-70, such as between 25-70, such asbetween 30-70, such as between 40-70, such as between 50-70, such asbetween 60-70.

21. The process of any of paragraphs 1-20, wherein the alpha-amylase isselected from the group of Bacillus stearothermophilus alpha-amylasevariants with the following mutations in addition to I181*+G182*, andoptionally N193F:

-   -   V59A+Q89R+G112D+E129V+K177L+R179E+K220P+N224L+Q254S;    -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;    -   V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+D269E+D281N;    -   V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+1270L;    -   V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+H274K;    -   V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+Y276F;    -   V59A+E129V+R157Y+K177L+R179E+K220P+N224L+S242Q+Q254S;    -   V59A+E129V+K177L+R179E+H208Y+K220P+N224L+S242Q+Q254S;    -   59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+H274K;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+Y276F;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+D281N;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+M284T;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+G416V;    -   V59A+E129V+K177L+R179E+K220P+N224L+Q254S;    -   V59A+E129V+K177L+R179E+K220P+N224L+Q254S+M284T;    -   A91L+M961+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;    -   E129V+K177L+R179E;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+Y276F+L427M;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+M284T;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+N376*+1377*;    -   E129V+K177L+R179E+K220P+N224L+Q254S;    -   E129V+K177L+R179E+K220P+N224L+Q254S+M284T;    -   E129V+K177L+R179E+S242Q;    -   E129V+K177L+R179V+K220P+N224L+S242Q+Q254S;    -   K220P+N224L+S242Q+Q254S;    -   M284V;    -   V59A Q89R+E129V+K177L+R179E+Q254S+M284V.

22. The process of any of paragraphs 1-21, wherein the alpha-amylase isselected from the group of Bacillus stearothermophilus alpha-amylasevariants:

-   -   I181*+G182*+N193F+E129V+K177L+R179E;    -   I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S    -   I181*+G182*+N193F+V59A Q89R+E129V+K177L+R179E+Q254S+M284V; and    -   I181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S        (using SEQ ID NO: 1 for numbering).

23. The process of any of paragraphs 1-22, wherein the alpha-amylasevariant has at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 91%, more preferably at least92%, even more preferably at least 93%, most preferably at least 94%,and even most preferably at least 95%, such as even at least 96%, atleast 97%, at least 98%, at least 99%, but less than 100% identity tothe mature part of the polypeptide of SEQ ID NO: 1 herein.

24. The process of any of paragraphs 1-23, wherein the alpha-amylase isa Bacillus licheniformis alpha-amylase, or a variant thereof.

25. The process of paragraph 24, wherein the Bacillus licheniformisalpha-amylase is the one shown in SEQ ID NO: 21 herein.

26. The process of any of paragraphs 1-25, wherein the alpha-amylase hasat least 80%, more preferably at least 85%, more preferably at least90%, more preferably at least 91%, more preferably at least 92%, evenmore preferably at least 93%, most preferably at least 94%, and evenmost preferably at least 95%, such as even at least 96%, at least 97%,at least 98%, at least 99% identity to the mature part of thepolypeptide of SEQ ID NO: 21 herein.

27. The process of any of paragraphs 1-26, wherein the alpha-amylase ispresent and/or added in a concentration of 0.1-100 micro gram per gramDS, such as 0.5-50 micro gram per gram DS, such as 1-25 micro gram pergram DS, such as 1-10 micro gram per gram DS, such as 2-5 micro gram pergram DS.

28. The process of any of paragraphs 1-27, wherein from 1-10 micro gramPyrococcus furiosus protease and 1-10 micro gram Bacillusstearothermophilus alpha-amylase are present and/or added inliquefaction.

29. The process of any of paragraphs 1-28, wherein a glucoamylase ispresent and/or added in liquefaction step a).

30. The process of paragraph 29, wherein the glucoamylase present and/oradded in liquefaction has a heat stability at 85° C., pH 5.3, of atleast 20%, such as at least 30%, preferably at least 35%.

31. The process of paragraph 29 or 30, wherein the glucoamylase has arelative activity pH optimum at pH 5.0 of at least 90%, preferably atleast 95%, preferably at least 97%.

32. The process of any of paragraphs 29-30, wherein the glucoamylase hasa pH stability at pH 5.0 of at least at least 80%, at least 85%, atleast 90%.

33. The process of any of paragraphs 29-32, wherein the glucoamylasepresent and/or added in liquefaction step a) is derived from a strain ofthe genus Penicillium, especially a strain of Penicillium oxalicumdisclosed as SEQ ID NO: 2 in WO 2011/127802 or SEQ ID NOs: 9 or 14herein.

34. The process of paragraph 29-33, wherein the glucoamylase has atleast 80%, more preferably at least 85%, more preferably at least 90%,more preferably at least 91%, more preferably at least 92%, even morepreferably at least 93%, most preferably at least 94%, and even mostpreferably at least 95%, such as even at least 96%, at least 97%, atleast 98%, at least 99% or 100% identity to the mature polypeptide shownin SEQ ID NO: 2 in WO 2011/127802 or SEQ ID NOs: 9 or 14 herein.

35. The process of any of paragraphs 29-34, wherein the glucoamylase isa variant of the Penicillium oxalicum glucoamylase shown in SEQ ID NO: 2in WO 2011/127802 having a K79V substitution (using the mature sequenceshown in SEQ ID NO: 14 for numbering), such as a variant disclosed in WO2013/053801.

36. The process of any of paragraph 29-35, wherein the Penicilliumoxalicum glucoamylase has a K79V substitution (using SEQ ID NO: 14 fornumbering) and further one of the following:

T65A; or

Q327F; or

E501V; or

Y504T; or

Y504*; or

T65A+Q327F; or

T65A+E501V; or

T65A+Y504T; or

T65A+Y504*; or

Q327F+E501V; or

Q327F+Y504T; or

Q327F+Y504*; or

E501V+Y504T; or

E501V+Y504*; or

T65A+Q327F+E501V; or

T65A+Q327F+Y504T; or

T65A+E501V+Y504T; or

Q327F+E501V+Y504T; or

T65A+Q327F+Y504*; or

T65A+E501V+Y504*; or

Q327F+E501V+Y504*; or

T65A+Q327F+E501V+Y504T; or

T65A+Q327F+E501V+Y504*;

E501V+Y504T; or

T65A+K161S; or

T65A+Q405T; or

T65A+Q327W; or

T65A+Q327F; or

T65A+Q327Y; or

P11F+T65A+Q327F; or

R1K+D3W+K5Q+G7V+N8S+T10K+P11S+T65A+Q327F; or

P2N+P4S+P11F+T65A+Q327F; or

P11F+D26C+K330+T65A+Q327F; or

P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; or

R1E+D3N+P4G+G6R+G7A+N8A+T10D+P11D+T65A+Q327F; or

P11F+T65A+Q327W; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or

P11F+T65A+Q327W+E501V+Y504T; or

T65A+Q327F+E501V+Y504T; or

T65A+S105P+Q327W; or

T65A+S105P+Q327F; or

T65A+Q327W+S364P; or

T65A+Q327F+S364P; or

T65A+S103N+Q327F; or

P2N+P4S+P11F+K34Y+T65A+Q327F; or

P2N+P4S+P11F+T65A+Q327F+D445N+V447S; or

P2N+P4S+P11F+T65A+I172V+Q327F; or

P2N+P4S+P11F+T65A+Q327F+N502*; or

P2N+P4S+P11F+T65A+Q327F+N502T+P563S+K571E; or

P2N+P4S+P11F+R31S+K33V+T65A+Q327F+N564D+K571S; or

P2N+P4S+P11F+T65A+Q327F+S377T; or

P2N+P4S+P11F+T65A+V325T+Q327W; or

P2N+P4S+P11F+T65A+Q327F+D445N+V447S+E501V+Y504T; or

P2N+P4S+P11F+T65A+1172V+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+S377T+E501V+Y504T; or

P2N+P4S+P11F+D26N+K34Y+T65A+Q327F; or

P2N+P4S+P11F+T65A+Q327F+I375A+E501V+Y504T; or

P2N+P4S+P11F+T65A+K218A+K221D+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+S103N+Q327F+E501V+Y504T; or

P2N+P4S+T10D+T65A+Q327F+E501V+Y504T; or

P2N+P4S+F12Y+T65A+Q327F+E501V+Y504T; or

K5A+P11F+T65A+Q327F+E501V+Y504T; or

P2N+P4S+T10E+E18N+T65A+Q327F+E501V+Y504T; or

P2N+T10E+E18N+T65A+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T568N; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+K524T+G526A; or

P2N+P4S+P11F+K34Y+T65A+Q327F+D445N+V447S+E501V+Y504T; or

P2N+P4S+P11F+R31S+K33V+T65A+Q327F+D445N+V447S+E501V+Y504T; or

P2N+P4S+P11F+D26N+K34Y+T65A+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+F80*+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+K112S+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T516P+K524T+G526A; or

P2N+P4S+P11F+T65A+Q327F+E501V+N502T+Y504*; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+S103N+Q327F+E501V+Y504T; or

K5A+P11F+T65A+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T516P+K524T+G526A; or

P2N+P4S+P11F+T65A+K79A+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+K79G+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+K791+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+K79L+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+K79S+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+L72V+Q327F+E501V+Y504T; or

S255N+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+E74N+V79K+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+G220N+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Y245N+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q253N+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+D279N+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+S359N+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+D370N+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+V460S+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+V460T+P468T+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+T463N+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+S465N+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+T477N+E501V+Y504T.

37. The process of any of paragraphs 29-36, wherein the glucoamylasepresent and/or added in liquefaction is the Penicillium oxalicumglucoamylase having a K79V substitution (using SEQ ID NO: 14 fornumbering) and further one of the following:

-   -   P11F+T65A+Q327F;    -   P2N+P4S+P11F+T65A+Q327F (using SEQ ID NO: 14 for numbering).

38. The process of any of paragraphs 11-27, wherein the glucoamylasevariant has at least 75% identity preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, more preferablyat least 91%, more preferably at least 92%, even more preferably atleast 93%, most preferably at least 94%, and even most preferably atleast 95%, such as even at least 96%, at least 97%, at least 98%, atleast 99%, but less than 100% identity to the mature part of thepolypeptide of SEQ ID NO: 14 herein.

39. The process of any of paragraphs 1-38, further wherein aglucoamylase is present and/or added in saccharification and/orfermentation.

40. The process of paragraph 39, wherein the glucoamylase present and/oradded in saccharification and/or fermentation is of fungal origin,preferably from a stain of Aspergillus, preferably A. niger, A. awamori,or A. oryzae; or a strain of Trichoderma, preferably T. reesei; or astrain of Talaromyces, preferably T. emersonii, or a strain ofPycnoporus, or a strain of Gloephyllum, such as G. serpiarium or G.trabeum, or a strain of the Nigrofomes.

41. The process of any of paragraphs 39-40, wherein the glucoamylase isderived from Talaromyces emersonii, such as the one shown in SEQ ID NO:19 herein,

42. The process of any of paragraphs 39-41, wherein the glucoamylase isselected from the group consisting of:

(i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 19herein;

(ii) a glucoamylase comprising an amino acid sequence having at least60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to the mature polypeptide of SEQ ID NO: 19 herein.

43. The process of any of paragraphs 39-42, wherein the glucoamylase isderived from Gloephyllum serpiarium, such as the one shown in SEQ ID NO:15 herein.

44. The process of any of paragraphs 39-43, wherein the glucoamylase isselected from the group consisting of:

(i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 15herein;

(ii) a glucoamylase comprising an amino acid sequence having at least60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to the mature polypeptide of SEQ ID NO: 15 herein.

45. The process of any of paragraphs 39-44, wherein the glucoamylase isderived from Gloeophyllum trabeum such as the one shown in SEQ ID NO: 17herein.

46. The process of any of paragraphs 39-45, wherein the glucoamylase isselected from the group consisting of:

(i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 17herein;

(ii) a glucoamylase comprising an amino acid sequence having at least60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to the mature polypeptide of SEQ ID NO: 17 herein.

47. The process of any of paragraphs 39-46, wherein the glucoamylase ispresent and/or added in saccharification and/or fermentation incombination with an alpha-amylase.

48. The process of paragraph 47, wherein the alpha-amylase presentand/or added in saccharification and/or fermentation is of fungal orbacterial origin.

49. The process of paragraph 47 or 48, wherein the alpha-amylase presentand/or added in saccharification and/or fermentation is derived from astrain of the genus Rhizomucor, preferably a strain the Rhizomucorpusillus, such as the one shown in SEQ ID NO: 3 in WO 2013/006756, suchas a Rhizomucor pusillus alpha-amylase hybrid having an Aspergillusniger linker and starch-bonding domain, such as the one shown in SEQ IDNO: 16 herein, or a variant thereof.

50. The process of any of paragraphs 47-49, wherein the alpha-amylasepresent and/or added in saccharification and/or fermentation is selectedfrom the group consisting of:

(i) an alpha-amylase comprising the mature polypeptide of SEQ ID NO: 16herein;

(ii) an alpha-amylase comprising an amino acid sequence having at least60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to the mature polypeptide of SEQ ID NO: 16 herein.

51. The process of any of paragraphs 47-50, wherein the alpha-amylase isa variant of the alpha-amylase shown in SEQ ID NO: 13 having at leastone of the following substitutions or combinations of substitutions:D165M; Y141W; Y141R; K136F; K192R; P224A; P224R; S123H+Y141W;G20S+Y141W; A76G+Y141W; G128D+Y141W; G128D+D143N; P219C+Y141W;N142D+D143N; Y141W+K192R; Y141W+D143N; Y141W+N383R; Y141W+P219C+A265C;Y141W+N142D+D143N; Y141W+K192R V410A; G128D+Y141W+D143N;Y141W+D143N+P219C; Y141W+D143N+K192R; G128D+D143N+K192R;Y141W+D143N+K192R+P219C; G128D+Y141W+D143N+K192R; orG128D+Y141W+D143N+K192R+P219C (using SEQ ID NO: 16 for numbering).

52. The process of any of paragraphs 47-51, wherein the alpha-amylase isderived from a Rhizomucor pusillus with an Aspergillus nigerglucoamylase linker and starch-binding domain (SBD), preferablydisclosed as SEQ ID NO: 13 herein, preferably having one or more of thefollowing substitutions: G128D, D143N, preferably G128D+D143N (using SEQID NO: 13 for numbering).

53. The process of amylase of paragraphs 47-52, wherein thealpha-amylase variant present and/or added in saccharification and/orfermentation has at least 75% identity preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, more preferablyat least 91%, more preferably at least 92%, even more preferably atleast 93%, most preferably at least 94%, and even most preferably atleast 95%, such as even at least 96%, at least 97%, at least 98%, atleast 99%, but less than 100% identity to the mature part of thepolypeptide of SEQ ID NO: 16 herein.

54. The process of any of paragraphs 1-53, further wherein a pullulanaseis present and/or added in liquefaction and/or saccharification and/orfermentation.

55. The process of paragraph 54, wherein the pullulanase is a familyGH57 pullulanase, wherein the pullulanase preferably includes an X47domain as disclosed in WO 2011/087836.

56. The process of paragraphs 54-55, wherein the pullulanase is derivedfrom a strain from the genus Thermococcus, including Thermococcuslitoralis and Thermococcus hydrothermalis or a hybrid thereof.

57. The process of any of paragraphs 54-56, wherein the pullulanase isthe truncated Thermococcus hydrothermalis pullulanase at site X4 or a T.hydrothermalis/T. litoralis hybrid enzyme with truncation site X4disclosed in WO 2011/087836 or shown in SEQ ID NO: 12 herein.

58. The process of any of paragraphs 1-57, further comprises, prior tothe liquefaction step a), the steps of:

-   -   i) reducing the particle size of the starch-containing material,        preferably by dry milling;    -   ii) forming a slurry comprising the starch-containing material        and water.

59. The process of any of paragraphs 1-58, wherein at least 50%,preferably at least 70%, more preferably at least 80%, especially atleast 90% of the starch-containing material fit through a sieve with #6screen.

60. The process of any of paragraphs 1-59, wherein the pH duringliquefaction is between above 4.5-6.5, such as around 4.8, or a pHbetween 5.0-6.2, such as 5.0-6.0, such as between 5.0-5.5, such asaround 5.2, such as around 5.4, such as around 5.6, such as around 5.8.

61. The process of any of paragraphs 1-60, wherein the temperatureduring liquefaction is above the initial gelatinization temperature,preferably in the range from 70-100° C., such as between 75-95° C., suchas between 75-90° C., preferably between 80-90° C., especially around85° C.

62. The process of any of paragraphs 1-61, wherein a jet-cooking step iscarried out before liquefaction in step a).

63. The process of paragraph 62, wherein the jet-cooking is carried outat a temperature between 110-145° C., preferably 120-140° C., such as125-135° C., preferably around 130° C. for about 1-15 minutes,preferably for about 3-10 minutes, especially around about 5 minutes.

64. The process of any of paragraphs 1-63, wherein saccharification andfermentation is carried out sequentially or simultaneously.

65. The process of any of paragraphs 1-64, wherein saccharification iscarried out at a temperature from 20-75° C., preferably from 40-70° C.,such as around 60° C., and at a pH between 4 and 5.

66. The process of any of paragraphs 1-65, wherein fermentation orsimultaneous saccharification and fermentation (SSF) is carried outcarried out at a temperature from 25° C. to 40° C., such as from 28° C.to 35° C., such as from 30° C. to 34° C., preferably around about 32° C.In an embodiment fermentation is ongoing for 6 to 120 hours, inparticular 24 to 96 hours.

67. The process of any of paragraphs 1-66, wherein the fermentationproduct is recovered after fermentation, such as by distillation.

68. The process of any of paragraphs 1-67, wherein the fermentationproduct is an alcohol, preferably ethanol, especially fuel ethanol,potable ethanol and/or industrial ethanol.

69. The process of any of paragraphs 1-68, wherein the starch-containingstarting material is whole grains.

70. The process of any of paragraphs 1-69, wherein the starch-containingmaterial is selected from the group of corn, wheat, barley, rye, milo,sago, cassava, manioc, tapioca, sorghum, rice, and potatoes.

71. The process of any of paragraphs 1-70, wherein the fermentingorganism is yeast, preferably a strain of Saccharomyces, especially astrain of Saccharomyces cerevisae.

72. The process of any of paragraphs 1-71, wherein the alpha-amylase isa bacterial or fungal alpha-amylase.

73. The process of any of paragraphs 1-72, wherein saccharification stepb) and fermentation step c) are carried out simultaneously orsequentially.

74. The process of any of paragraphs 1-73, wherein the temperature instep (a) is above the initial gelatinization temperature, such as at atemperature between 80-90° C., such as around 85° C.

75. The process of any of paragraphs 1-74, further comprising apre-saccharification step, before saccharification step b), carried outfor 40-90 minutes at a temperature between 30-65° C.

76. The process of any of paragraphs 1-75, wherein saccharification iscarried out at a temperature from 20-75° C., preferably from 40-70° C.,such as around 60° C., and at a pH between 4 and 5.

77. The process of any of paragraphs 1-76, wherein fermentation step c)or simultaneous saccharification and fermentation (SSF) (i.e., steps b)and c)) are carried out carried out at a temperature from 25° C. to 40°C., such as from 28° C. to 35° C., such as from 30° C. to 34° C.,preferably around about 32° C.

78. The process of any of paragraphs 1-77, wherein fermentation step c)or simultaneous saccharification and fermentation (SSF) (i.e., steps b)and c)) are ongoing for 6 to 120 hours, in particular 24 to 96 hours.

79. The process of any of paragraphs 1-78, wherein separation in step e)is carried out by centrifugation, preferably a decanter centrifuge,filtration, preferably using a filter press, a screw press, aplate-and-frame press, a gravity thickener or decker.

80. The process of any of paragraphs 1-79, wherein the fermentationproduct is recovered by distillation.

81. A process of recovering oil of any of paragraphs 1-80, comprisingthe steps of:

a) liquefying starch-containing material at a temperature above theinitial gelatinization temperature using:

-   -   Bacillus stearothermophilus alpha-amylase comprising a double        deletion at positions I181+G182 using SEQ ID NO: 1 for        numbering;    -   more than 0.5 micro gram Pyrococcus furiosus protease per gram        dry solids (DS);    -   Penicillium oxalicum shown in SEQ ID NO: 14 comprising a K79V        substitution;

b) saccharifying using a glucoamylase;

c) fermenting using a fermenting organism.

d) recovering the fermentation product to form whole stillage;

e) separating the whole stillage into thin stillage and wet cake;

f) optionally concentrating the thin stillage into syrup;

wherein oil is recovered from the:

-   -   liquefied starch-containing material after step a); and/or    -   downstream from fermentation step c).

82. A process of recovering oil of any of paragraphs 1-81 comprising thesteps of:

a) liquefying starch-containing material at a temperature above theinitial gelatinization temperature using:

-   -   Bacillus stearothermophilus alpha-amylase comprising a double        deletion at positions I181+G182 and the following substitutions        N193F+V59A+Q89R+E129V+K177L+R179E+Q254S+M284V truncated to 491        amino acids (using SEQ ID NO: 1 for numbering).    -   more than 0.5 micro gram Pyrococcus furiosus protease per gram        dry solids (DS);    -   Penicillium oxalicum glucoamylase having the following        mutations: K79V+P2N+P4S+P11F+T65A+Q327F (using SEQ ID NO: 14 for        numbering);

b) saccharifying using a glucoamylase;

c) fermenting using a fermenting organism.

d) recovering the fermentation product to form whole stillage;

e) separating the whole stillage into thin stillage and wet cake;

f) optionally concentrating the thin stillage into syrup;

wherein oil is recovered from the:

-   -   liquefied starch-containing material after step a); and/or    -   downstream from fermentation step c).

83. The process of any of paragraphs 1-82, wherein the ratio betweenalpha-amylase and glucoamylase in liquefaction is between 1:1 and 1:10,such as around 1:2 (micro gram alpha-amylase per gram DS:micro gramglucoamylase per gram DS).

84. The process of any of paragraphs 1-83, wherein the ratio betweenalpha-amylase and protease in liquefaction is in the range between 1:1and 1:25, such between 1:1.2 and as 1:10, such as around 1:1.4 (microgram alpha-amylase per gram DS:micro gram protease per gram DS).

85. A process for producing fermentation products from starch-containingmaterial comprising the steps of:

-   -   a) liquefying the starch-containing material at a temperature        above the initial gelatinization temperature using:        -   an alpha-amylase;        -   more than 2 micro gram Pyrococcus furiosus protease per gram            dry solids (DS);    -   b) saccharifying using a glucoamylase;    -   c) fermenting using a fermenting organism.

86. The process of paragraph 85, wherein 2-100 micro gram per gram DS,such as 2.5-50 micro gram per gram DS, such as 2.5-10 micro gram pergram DS, such as 2.5-5 micro gram per gram DS, especially around 3 microgram per gram DS Pyrococcus furiosus protease.

87. The process of any of paragraph 85 or 86, wherein the Pyrococcusfuriosus protease is the one shown in SEQ ID NO: 13 herein.

88. The process of any of paragraphs 85-87, wherein the Pyrococcusfuriosus protease is one having at least 80%, such as at least 85%, suchas at least 90%, such as at least 95%, such as at least 96%, such as atleast 97%, such as at least 98%, such as at least 99% identity to SEQ IDNO: 13 herein.

89. The process of any of paragraphs 85-88, wherein no nitrogen-compoundis present and/or added in steps a)-c), such as during saccharificationstep b) or fermentation step c) or simultaneous saccharification andfermentation (SSF).

90. The process of any of paragraphs 85-89, wherein 10-1,000 ppm, suchas 50-800 ppm, such as 100-600 ppm, such as 200-500 ppmnitrogen-compound, preferably urea, is present and/or added in stepsa)-c), such as during saccharification step b) or fermentation step c)or simultaneous saccharification and fermentation (SSF).

91. The process of any of paragraphs 85-90, wherein the alpha-amylase isfrom the genus Bacillus, such as a strain of Bacillusstearothermophilus, in particular a variant of a Bacillusstearothermophilus alpha-amylase, such as the one shown in SEQ ID NO: 3in WO 99/019467 or SEQ ID NO: 1 herein, or a variant thereof or a strainof Bacillus licheniformis, such as the one shown in SEQ ID NO: 21herein.

92. The process of paragraph 91, wherein the Bacillus stearothermophilusalpha-amylase or variant thereof is truncated, preferably to have around491 amino acids, such as from 480-495 amino acids.

93. The process of any of paragraphs 91 or 92, wherein the Bacillusstearothermophilus alpha-amylase has a double deletion at positionsI181+G182 and optionally a N193F substitution, or deletion of R179 andG180 (using SEQ ID NO: 1 for numbering).

94. The process of any of paragraphs 85-93 wherein the Bacillusstearothermophilus alpha-amylase has a substitution at position S242,preferably S242Q substitution.

95. The process of any of paragraphs 85-94, wherein the Bacillusstearothermophilus alpha-amylase has a substitution at position E188,preferably E188P substitution.

96. The process of any of paragraphs 85-95, wherein the alpha-amylasehas a T½ (min) at pH 4.5, 85° C., 0.12 mM CaCl₂) of at least 10, such asat least 15, such as at least 20, such as at least 25, such as at least30, such as at least 40, such as at least 50, such as at least 60, suchas between 10-70, such as between 15-70, such as between 20-70, such asbetween 25-70, such as between 30-70, such as between 40-70, such asbetween 50-70, such as between 60-70.

97. The process of any of paragraphs 85-96, wherein the alpha-amylase isselected from the group of Bacillus stearothermophilus alpha-amylasevariants with the following mutations in addition to I181*+G182* andoptionally N193F:

-   -   V59A+Q89R+G112D+E129V+K177L+R179E+K220P+N224L+Q254S;    -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;    -   V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+D269E+D281N;    -   V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+1270L;    -   V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+H274K;    -   V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+Y276F;    -   V59A+E129V+R157Y+K177L+R179E+K220P+N224L+S242Q+Q254S;    -   V59A+E129V+K177L+R179E+H208Y+K220P+N224L+S242Q+Q254S;    -   59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+H274K;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+Y276F;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+D281N;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+M284T;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+G416V;    -   V59A+E129V+K177L+R179E+K220P+N224L+Q254S;    -   V59A+E129V+K177L+R179E+K220P+N224L+Q254S+M284T;    -   A91L+M961+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;    -   E129V+K177L+R179E;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+Y276F+L427M;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+M284T;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+N376*+1377*;    -   E129V+K177L+R179E+K220P+N224L+Q254S;    -   E129V+K177L+R179E+K220P+N224L+Q254S+M284T;    -   E129V+K177L+R179E+S242Q;    -   E129V+K177L+R179V+K220P+N224L+S242Q+Q254S;    -   K220P+N224L+S242Q+Q254S;    -   M284V;    -   V59A Q89R+E129V+K177L+R179E+Q254S+M284V.

98. The process of any of paragraphs 85-97, wherein the alpha-amylase isselected from the group of Bacillus stearothermophilus alpha-amylasevariants:

-   -   I181*+G182*+N193F+E129V+K177L+R179E;    -   I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S    -   I181*+G182*+N193F+V59A Q89R+E129V+K177L+R179E+Q254S+M284V; and    -   I181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S        (using SEQ ID NO: 1 for numbering).

99. The process of any of paragraphs 91-98, wherein the alpha-amylasevariant has at least 75% identity preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, more preferablyat least 91%, more preferably at least 92%, even more preferably atleast 93%, most preferably at least 94%, and even most preferably atleast 95%, such as even at least 96%, at least 97%, at least 98%, atleast 99%, but less than 100% identity to the mature part of thepolypeptide of SEQ ID NO: 1 herein.

100. The process of any of paragraphs 85-98, wherein the alpha-amylaseis a Bacillus licheniformis alpha-amylase, or a variant thereof.

101. The process of paragraph 100, wherein the Bacillus licheniformisalpha-amylase is the one shown in SEQ ID NO: 21 herein.

102. The process of any of paragraphs 100-101, wherein the alpha-amylasehas at least 80%, more preferably at least 85%, more preferably at least90%, more preferably at least 91%, more preferably at least 92%, evenmore preferably at least 93%, most preferably at least 94%, and evenmost preferably at least 95%, such as even at least 96%, at least 97%,at least 98%, at least 99% identity to the mature part of thepolypeptide of SEQ ID NO: 21 herein.

103. The process of any of paragraphs 85-102, wherein the alpha-amylaseis present and/or added in a concentration of 0.1-100 micro gram pergram DS, such as 0.5-50 micro gram per gram DS, such as 1-25 micro gramper gram DS, such as 1-10 micro gram per gram DS, such as 2-5 micro gramper gram DS.

104. The process of any of paragraphs 85-103, wherein from 1-10 microgram Pyrococcus furiosus protease and 1-10 micro gram Bacillusstearothermophilus alpha-amylase are present and/or added inliquefaction.

105. The process of any of paragraphs 85-104, wherein a glucoamylase ispresent and/or added in liquefaction step i).

106. The process of paragraph 105, wherein the glucoamylase presentand/or added in liquefaction has a heat stability at 85° C., pH 5.3, ofat least 20%, such as at least 30%, preferably at least 35%.

107. The process of paragraph 105 or 106, wherein the glucoamylase has arelative activity pH optimum at pH 5.0 of at least 90%, preferably atleast 95%, preferably at least 97%.

108. The process of any of paragraphs 105-102, wherein the glucoamylasehas a pH stability at pH 5.0 of at least at least 80%, at least 85%, atleast 90%.

109. The process of any of paragraphs 105-103, wherein the glucoamylasepresent and/or added in liquefaction step i) is derived from a strain ofthe genus Penicillium, especially a strain of Penicillium oxalicumdisclosed as SEQ ID NO: 2 in WO 2011/127802 or SEQ ID NOs: 9 or 14herein.

110. The process of paragraph 105-109, wherein the glucoamylase has atleast 80%, more preferably at least 85%, more preferably at least 90%,more preferably at least 91%, more preferably at least 92%, even morepreferably at least 93%, most preferably at least 94%, and even mostpreferably at least 95%, such as even at least 96%, at least 97%, atleast 98%, at least 99% or 100% identity to the mature polypeptide shownin SEQ ID NO: 2 in WO 2011/127802 or SEQ ID NOs: 9 or 14 herein.

111. The process of any of paragraphs 105-110, wherein the glucoamylaseis a variant of the Penicillium oxalicum glucoamylase shown in SEQ IDNO: 2 in WO 2011/127802 having a K79V substitution (using the maturesequence shown in SEQ ID NO: 14 for numbering), such as a variantdisclosed in WO 2013/053801.

112. The process of any of paragraph 105-111, wherein the Penicilliumoxalicum glucoamylase has a K79V substitution (using SEQ ID NO: 14 fornumbering) and further one of the following:

T65A; or

Q327F; or

E501V; or

Y504T; or

Y504*; or

T65A+Q327F; or

T65A+E501V; or

T65A+Y504T; or

T65A+Y504*; or

Q327F+E501V; or

Q327F+Y504T; or

Q327F+Y504*; or

E501V+Y504T; or

E501V+Y504*; or

T65A+Q327F+E501V; or

T65A+Q327F+Y504T; or

T65A+E501V+Y504T; or

Q327F+E501V+Y504T; or

T65A+Q327F+Y504*; or

T65A+E501V+Y504*; or

Q327F+E501V+Y504*; or

T65A+Q327F+E501V+Y504T; or

T65A+Q327F+E501V+Y504*;

E501V+Y504T; or

T65A+K161S; or

T65A+Q405T; or

T65A+Q327W; or

T65A+Q327F; or

T65A+Q327Y; or

P11F+T65A+Q327F; or

R1K+D3W+K5Q+G7V+N8S+T10K+P11S+T65A+Q327F; or

P2N+P4S+P11F+T65A+Q327F; or

P11F+D26C+K330+T65A+Q327F; or

P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; or

R1E+D3N+P4G+G6R+G7A+N8A+T10D+P11D+T65A+Q327F; or

P11F+T65A+Q327W; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or

P11F+T65A+Q327W+E501V+Y504T; or

T65A+Q327F+E501V+Y504T; or

T65A+S105P+Q327W; or

T65A+S105P+Q327F; or

T65A+Q327W+S364P; or

T65A+Q327F+S364P; or

T65A+S103N+Q327F; or

P2N+P4S+P11F+K34Y+T65A+Q327F; or

P2N+P4S+P11F+T65A+Q327F+D445N+V447S; or

P2N+P4S+P11F+T65A+I172V+Q327F; or

P2N+P4S+P11F+T65A+Q327F+N502*; or

P2N+P4S+P11F+T65A+Q327F+N502T+P563S+K571E; or

P2N+P4S+P11F+R31S+K33V+T65A+Q327F+N564D+K571S; or

P2N+P4S+P11F+T65A+Q327F+S377T; or

P2N+P4S+P11F+T65A+V325T+Q327W; or

P2N+P4S+P11F+T65A+Q327F+D445N+V447S+E501V+Y504T; or

P2N+P4S+P11F+T65A+I172V+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+S377T+E501V+Y504T; or

P2N+P4S+P11F+D26N+K34Y+T65A+Q327F; or

P2N+P4S+P11F+T65A+Q327F+I375A+E501V+Y504T; or

P2N+P4S+P11F+T65A+K218A+K221D+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+S103N+Q327F+E501V+Y504T; or

P2N+P4S+T10D+T65A+Q327F+E501V+Y504T; or

P2N+P4S+F12Y+T65A+Q327F+E501V+Y504T; or

K5A+P11F+T65A+Q327F+E501V+Y504T; or

P2N+P4S+T10E+E18N+T65A+Q327F+E501V+Y504T; or

P2N+T10E+E18N+T65A+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T568N; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+K524T+G526A; or

P2N+P4S+P11F+K34Y+T65A+Q327F+D445N+V447S+E501V+Y504T; or

P2N+P4S+P11F+R31S+K33V+T65A+Q327F+D445N+V447S+E501V+Y504T; or

P2N+P4S+P11F+D26N+K34Y+T65A+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+F80*+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+K112S+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T516P+K524T+G526A; or

P2N+P4S+P11F+T65A+Q327F+E501V+N502T+Y504*; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+S103N+Q327F+E501V+Y504T; or

K5A+P11F+T65A+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T516P+K524T+G526A; or

P2N+P4S+P11F+T65A+K79A+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+K79G+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+K791+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+K79L+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+K79S+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+L72V+Q327F+E501V+Y504T; or

S255N+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+E74N+V79K+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+G220N+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Y245N+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q253N+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+D279N+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+S359N+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+D370N+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+V460S+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+V460T+P468T+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+T463N+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+S465N+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+T477N+E501V+Y504T.

113. The process of any of paragraphs 105-112, wherein the glucoamylasepresent and/or added in liquefaction is the Penicillium oxalicumglucoamylase having a K79V substitution (using SEQ ID NO: 14 fornumbering) and further one of the following:

-   -   P11F T65A Q327F    -   P2N P4S P11F T65A Q327F (using SEQ ID NO: 14 for numbering).

114. The process of any of paragraphs 105-113, wherein the glucoamylasevariant has at least 75% identity preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, more preferablyat least 91%, more preferably at least 92%, even more preferably atleast 93%, most preferably at least 94%, and even most preferably atleast 95%, such as even at least 96%, at least 97%, at least 98%, atleast 99%, but less than 100% identity to the mature part of thepolypeptide of SEQ ID NO: 14 herein.

115. The process of any of paragraphs 85-114, further wherein aglucoamylase is present and/or added in saccharification and/orfermentation.

116. The process of paragraph 115, wherein the glucoamylase presentand/or added in saccharification and/or fermentation is of fungalorigin, preferably from a stain of Aspergillus, preferably A. niger, A.awamori, or A. oryzae; or a strain of Trichoderma, preferably T. reesei;or a strain of Talaromyces, preferably T. emersonii, or a strain ofPycnoporus, or a strain of Gloephyllum, such as G. serpiarium or G.trabeum, or a strain of the Nigrofomes.

117. The process of any of paragraphs 115-116, wherein the glucoamylaseis derived from Talaromyces emersonii, such as the one shown in SEQ IDNO: 19 herein,

118. The process of any of paragraphs 115-117, wherein the glucoamylaseis selected from the group consisting of:

(i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 19herein;

(ii) a glucoamylase comprising an amino acid sequence having at least60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to the mature polypeptide of SEQ ID NO: 19 herein.

119. The process of any of paragraphs 115-119, wherein the glucoamylaseis derived from Gloephyllum serpiarium, such as the one shown in SEQ IDNO: 15 herein.

120. The process of any of paragraphs 115-119, wherein the glucoamylaseis selected from the group consisting of:

(i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 15herein;

(ii) a glucoamylase comprising an amino acid sequence having at least60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to the mature polypeptide of SEQ ID NO: 15 herein.

121. The process of any of paragraphs 115-120, wherein the glucoamylaseis derived from Gloeophyllum trabeum such as the one shown in SEQ ID NO:17 herein.

122. The process of any of paragraphs 115-121, wherein the glucoamylaseis selected from the group consisting of:

(i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 17herein;

(ii) a glucoamylase comprising an amino acid sequence having at least60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to the mature polypeptide of SEQ ID NO: 17 herein.

123. The process of any of paragraphs 115-122, wherein the glucoamylaseis present in saccharification and/or fermentation in combination withan alpha-amylase.

124. The process of paragraph 123, wherein the alpha-amylase is presentin saccharification and/or fermentation is of fungal or bacterialorigin.

125. The process of paragraph 123 or 124, wherein the alpha-amylasepresent and/or added in saccharification and/or fermentation is derivedfrom a strain of the genus Rhizomucor, preferably a strain theRhizomucor pusillus, such as the one shown in SEQ ID NO: 3 in WO2013/006756, such as a Rhizomucor pusillus alpha-amylase hybrid havingan Aspergillus niger linker and starch-bonding domain, such as the oneshown in SEQ ID NO: 16.

126. The process of any of paragraphs 123-125, wherein the alpha-amylasepresent in saccharification and/or fermentation is selected from thegroup consisting of:

(i) an alpha-amylase comprising the mature polypeptide of SEQ ID NO: 16herein;

(ii) an alpha-amylase comprising an amino acid sequence having at least60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to the mature polypeptide of SEQ ID NO: 16 herein.

127. The process of any of paragraphs 123-126, wherein the alpha-amylaseis a variant of the alpha-amylase shown in SEQ ID NO: 13 having at leastone of the following substitutions or combinations of substitutions:D165M; Y141W; Y141R; K136F; K192R; P224A; P224R; S123H+Y141W;G20S+Y141W; A76G+Y141W; G128D+Y141W; G128D+D143N; P219C+Y141W;N142D+D143N; Y141W+K192R; Y141W+D143N; Y141W+N383R; Y141W+P219C+A265C;Y141W+N142D+D143N; Y141W+K192R V410A; G128D+Y141W+D143N;Y141W+D143N+P219C; Y141W+D143N+K192R; G128D+D143N+K192R;Y141W+D143N+K192R+P219C; G128D+Y141W+D143N+K192R; orG128D+Y141W+D143N+K192R+P219C (using SEQ ID NO: 16 for numbering).

128. The process of any of paragraphs 123-127, wherein the alpha-amylaseis derived from a Rhizomucor pusillus with an Aspergillus nigerglucoamylase linker and starch-binding domain (SBD), preferablydisclosed as SEQ ID NO: 16 herein, preferably having one or more of thefollowing substitutions: G128D, D143N, preferably G128D+D143N (using SEQID NO: 16 for numbering).

129. The process of any of paragraphs 123-128, wherein the alpha-amylasevariant has at least 75% identity preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, more preferablyat least 91%, more preferably at least 92%, even more preferably atleast 93%, most preferably at least 94%, and even most preferably atleast 95%, such as even at least 96%, at least 97%, at least 98%, atleast 99%, but less than 100% identity to the mature part of thepolypeptide of SEQ ID NO: 16 herein.

130. The process of any of paragraphs 85-129, further wherein apullulanase is present and/or added in liquefaction and/orsaccharification and/or fermentation.

131. The process of paragraph 130, wherein the pullulanase is a familyGH57 pullulanase, wherein the pullulanase preferably includes an X47domain as disclosed in WO 2011/087836.

132. The process of paragraphs 130-131, wherein the pullulanase isderived from a strain from the genus Thermococcus, includingThermococcus litoralis and Thermococcus hydrothermalis or a hybridthereof.

133. The process of any of paragraphs 130-132, wherein the pullulanaseis the truncated Thermococcus hydrothermalis pullulanase at site X4 or aT. hydrothermalis/T. litoralis hybrid enzyme with truncation site X4disclosed in WO 2011/087836 or shown in SEQ ID NO: 12 herein.

134. The process of any of paragraphs 85-133, further comprises, priorto the liquefaction step i), the steps of:

-   -   i) reducing the particle size of the starch-containing material,        preferably by dry milling;    -   ii) forming a slurry comprising the starch-containing material        and water.

135. The process of any of paragraphs 85-134, wherein at least 50%,preferably at least 70%, more preferably at least 80%, especially atleast 90% of the starch-containing material fit through a sieve with #6screen.

136. The process of any of paragraphs 85-135, wherein the pH inliquefaction is between above 4.5-6.5, such as around 4.8, or a pHbetween 5.0-6.2, such as 5.0-6.0, such as between 5.0-5.5, such asaround 5.2, such as around 5.4, such as around 5.6, such as around 5.8.

137. The process of any of paragraphs 85-136, wherein the temperature inliquefaction is above the initial gelatinization temperature, such as inthe range from 70−100° C., such as between 75-95° C., such as between75-90° C., preferably between 80-90° C., especially around 85° C.

138. The process of any of paragraphs 85-137, wherein a jet-cooking stepis carried out before liquefaction in step a).

139. The process of paragraph 138, wherein the jet-cooking is carriedout at a temperature between 110-145° C., preferably 120-140° C., suchas 125-135° C., preferably around 130° C. for about 1-15 minutes,preferably for about 3-10 minutes, especially around about 5 minutes.

140. The process of any of paragraphs 85-139, wherein saccharificationand fermentation is carried out sequentially or simultaneously.

141. The process of any of paragraphs 85-140, wherein saccharificationis carried out at a temperature from 20-75° C., preferably from 40-70°C., such as around 60° C., and at a pH between 4 and 5.

142. The process of any of paragraphs 85-141, wherein fermentation orsimultaneous saccharification and fermentation (SSF) is carried outcarried out at a temperature from 25° C. to 40° C., such as from 28° C.to 35° C., such as from 30° C. to 34° C., preferably around about 32° C.In an embodiment fermentation is ongoing for 6 to 120 hours, inparticular 24 to 96 hours.

143. The process of any of paragraphs 85-142, wherein the fermentationproduct is recovered after fermentation, such as by distillation.

144. The process of any of paragraphs 85-143, wherein the fermentationproduct is an alcohol, preferably ethanol, especially fuel ethanol,potable ethanol and/or industrial ethanol.

145. The process of any of paragraphs 85-144, wherein thestarch-containing starting material is whole grains.

146. The process of any of paragraphs 85-145, wherein thestarch-containing material is derived from corn, wheat, barley, rye,milo, sago, cassava, manioc, tapioca, sorghum, rice or potatoes.

147. The process of any of paragraphs 85-146, wherein the fermentingorganism is yeast, preferably a strain of Saccharomyces, especially astrain of Saccharomyces cerevisae.

148. The process of any of paragraphs 85-147, wherein the alpha-amylaseis a bacterial or fungal alpha-amylase.

149. The process of any of paragraphs 85-148, comprising the steps of:

a) liquefying the starch-containing material at a temperature above theinitial gelatinization temperature using:

-   -   an alpha-amylase derived from Bacillus stearothermophilus;    -   more than 2 micro gram Pyrococcus furiosus protease per gram dry        solids (DS); and    -   optionally a Penicillium oxalicum glucoamylase;

b) saccharifying using a glucoamylase enzyme;

c) fermenting using a fermenting organism.

150. A process of paragraphs 85-149, comprising the steps of:

a) liquefying the starch-containing material at a temperature above theinitial gelatinization temperature using:

-   -   an alpha-amylase, preferably derived from Bacillus        stearothermophilus, having a T½ (min) at pH 4.5, 85° C., 0.12 mM        CaCl₂ of at least 10;    -   more than 2 micro gram Pyrococcus furiosus protease per gram dry        solids (DS); and    -   optionally a glucoamylase;

b) saccharifying using a glucoamylase enzyme;

c) fermenting using a fermenting organism.

151. A process of paragraphs 85-150, comprising the steps of:

a) liquefying the starch-containing material at a temperature above theinitial gelatinization temperature using:

-   -   an alpha-amylase, preferably derived from Bacillus        stearothermophilus, having a T½ (min) at pH 4.5, 85° C., 0.12 mM        CaCl₂) of at least 10;    -   more than 2 micro gram Pyrococcus furiosus protease per gram dry        solids (DS); and    -   a Penicillium oxalicum glucoamylasea;

b) saccharifying using a glucoamylase enzyme;

c) fermenting using a fermenting organism.

152. A process of paragraphs 85-151, comprising the steps of:

-   -   a) liquefying the starch-containing material at a temperature        above the initial gelatinization temperature using:        -   an alpha-amylase derived from Bacillus stearothermophilus            having a double deletion at positions I181+G182, and            optional substitution N193F; further one of the following            set of substitutions:            -   E129V+K177L+R179E;            -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;            -   V59A+Q89R+E129V+K177L+R179E+Q254S+M284V;            -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID                NO: 1 herein for numbering);        -   more than 2 micro gram Pyrococcus furiosus protease per gram            dry solids (DS);        -   a Penicillium oxalicum glucoamylase in SEQ ID NO: 14 having            substitutions selected from the group of:        -   K79V;        -   K79V+P11F+T65A+Q327F; or        -   K79V+P2N+P4S+P11F+T65A+Q327F; or        -   K79V+P11F+D26C+K33C+T65A+Q327F; or        -   K79V+P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; or        -   K79V+P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or        -   K79V+P11F+T65A+Q327W+E501V+Y504T (using SEQ ID NO: 14 for            numbering);    -   b) saccharifying using a glucoamylase enzyme;    -   c) fermenting using a fermenting organism.

153. A process of paragraphs 85-152, comprising the steps of:

-   -   a) liquefying the starch-containing material at a pH in the        range between from above 4.5-6.5 at a temperature between        80-90° C. using:        -   an alpha-amylase derived from Bacillus stearothermophilus            having a double deletion I181+G182 and optional substitution            N193F; and further one of the following set of            substitutions:            -   E129V+K177L+R179E;            -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;            -   V59A+Q89R+E129V+K177L+R179E+Q254S+M284V;            -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID                NO: 1 herein for numbering);        -   more than 2 micro gram, such as between 2-5 micro gram,            preferably around 3 micro gram Pyrococcus furiosus protease            per gram DS dry solids (DS);        -   a Penicillium oxalicum glucoamylase in SEQ ID NO: 14 having            substitutions selected from the group of:        -   K79V;        -   K79V+P11F+T65A+Q327F; or        -   K79V+P2N+P4S+P11F+T65A+Q327F; or        -   K79V+P11F+D26C+K33C+T65A+Q327F; or        -   K79V+P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; or        -   K79V+P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or        -   K79V+P11F+T65A+Q327W+E501V+Y504T (using SEQ ID NO: 14 for            numbering);    -   b) saccharifying using a glucoamylase enzyme;    -   c) fermenting using a fermenting organism.

154. A process of paragraphs 85-153, comprising the steps of:

-   -   a) liquefying the starch-containing material at a pH in the        range between from above 4.5-6.5 at a temperature between        80-90° C. using:        -   an alpha-amylase derived from Bacillus stearothermophilus            having a double deletion I181+G182 and substitution N193F;            and further one of the following set of substitutions:            -   E129V+K177L+R179E;            -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;            -   V59A+Q89R+E129V+K177L+R179E+Q254S+M284V;            -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID                NO: 1 herein for numbering);        -   more than 2 micro gram, such as between 2-5 micro gram,            preferably around 3 micro gram Pyrococcus furiosus protease            per gram DS dry solids (DS)        -   a Penicillium oxalicum glucoamylase in SEQ ID NO: 14 having            substitutions selected from the group of:        -   K79V;        -   K79V+P11F+T65A+Q327F; or        -   K79V+P2N+P4S+P11F+T65A+Q327F; or        -   K79V+P11F+D26C+K33C+T65A+Q327F; or        -   K79V+P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; or        -   K79V+P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or        -   K79V+P11F+T65A+Q327W+E501V+Y504T (using SEQ ID NO: 14 for            numbering);    -   b) saccharifying using a Rhizomucor pusillus glucoamylase with        an Aspergillus niger glucoamylase linker and starch-binding        domain (SBD), preferably disclosed as SEQ ID NO: 13 herein,        preferably having one or more of the following substitutions:        G128D, D143N, preferably G128D+D143N (using SEQ ID NO: 13 for        numbering);    -   c) fermenting using a fermenting organism.

155. A process of any of paragraphs 85-154, wherein the ratio betweenalpha-amylase and glucoamylase in liquefaction is between 1:1 and 1:10,such as around 1:2 (micro gram alpha-amylase per g DS:micro gramglucoamylase per gram DS).

156. A process of any of paragraphs 85-155, wherein the ratio betweenalpha-amylase and protease in liquefaction is in the range between 1:1and 1:25, such between 1:1.2 and as 1:10, such as around 1:1.4 (microgram alpha-amylase per g DS:micro gram protease per gram DS).

157. An enzyme composition comprising:

-   -   i) Bacillus sp. alpha-amylase, or a variant thereof;    -   ii) Pyrococcus furiosus protease;

wherein the ratio between alpha-amylase and protease is in the rangefrom 1:1 and 1:25 (micro gram alpha-amylase:micro gram protease).

158. The enzyme composition paragraph 157, wherein the ratio betweenalpha-amylase and protease is in the range between 1:1.2 and 1:10, suchas around 1:1.4 (micro gram alpha-amylase:micro gram protease).

159. The enzyme composition of any of paragraphs 157-158, wherein theenzyme composition comprises a glucoamylase and the ratio betweenalpha-amylase and glucoamylase in liquefaction is between 1:1 and 1:10,such as around 1:2 (micro gram alpha-amylase:micro gram glucoamylase).

160. The enzyme composition of any of paragraphs 157-159, wherein thealpha-amylase is a bacterial or fungal alpha-amylase.

161, The enzyme composition of any of paragraphs 157-160, wherein thealpha-amylase is from the genus Bacillus, such as a strain of Bacillusstearothermophilus, in particular a variant of a Bacillusstearothermophilus alpha-amylase, such as the one shown in SEQ ID NO: 3in WO 99/019467 or SEQ ID NO: 1 herein.

162. The enzyme composition of any of paragraphs 157-161, wherein theBacillus stearothermophilus alpha-amylase or variant thereof istruncated, preferably to have around 491 amino acids, such as from480-495 amino acids.

163. The enzyme composition of any of paragraphs 157-162, wherein theBacillus stearothermophilus alpha-amylase has a double deletion,preferably at positions I181+G182 and optionally a N193F substitution,or double deletion of R179 and G180 (using SEQ ID NO: 1 for numbering).

164. The enzyme composition of any of paragraphs 157-163 wherein theBacillus stearothermophilus alpha-amylase has a substitution at positionS242, preferably S242Q substitution.

165. The enzyme composition of any of paragraphs 157-164, wherein theBacillus stearothermophilus alpha-amylase has a substitution at positionE188, preferably E188P substitution.

166. The enzyme composition of any of paragraphs 157-165, wherein thealpha-amylase has a T½ (min) at pH 4.5, 85° C., 0.12 mM CaCl₂) of atleast 10, such as at least 15, such as at least 20, such as at least 25,such as at least 30, such as at least 40, such as at least 50, such asat least 60, such as between 10-70, such as between 15-70, such asbetween 20-70, such as between 25-70, such as between 30-70, such asbetween 40-70, such as between 50-70, such as between 60-70.

167. The enzyme composition of any of paragraphs 157-166, wherein thealpha-amylase is selected from the group of Bacillus stearomthermphilusalpha-amylase variants with the following mutations:

-   -   I181*+G182*+N193F+E129V+K177L+R179E;    -   I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;    -   I181*+G182*+N193F+V59A Q89R+E129V+K177L+R179E+Q254S+M284V; and    -   I181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S        (using SEQ ID NO: 1 herein for numbering).

168. The enzyme composition of any of paragraphs 157-167, wherein thealpha-amylase variant has at least 75% identity preferably at least 80%,more preferably at least 85%, more preferably at least 90%, morepreferably at least 91%, more preferably at least 92%, even morepreferably at least 93%, most preferably at least 94%, and even mostpreferably at least 95%, such as even at least 96%, at least 97%, atleast 98%, at least 99%, but less than 100% identity to the mature partof the polypeptide of SEQ ID NO: 1 herein.

169. The enzyme composition of any of paragraphs 157-168, wherein thealpha-amylase is a Bacillus licheniformis alpha-amylase, or a variantthereof.

170. The enzyme composition of paragraph 169, wherein the Bacilluslicheniformis alpha-amylase is the one shown in SEQ ID NO: 21 herein.

171. The enzyme composition of any of paragraphs 157-170, wherein thealpha-amylase has at least 80%, more preferably at least 85%, morepreferably at least 90%, more preferably at least 91%, more preferablyat least 92%, even more preferably at least 93%, most preferably atleast 94%, and even most preferably at least 95%, such as even at least96%, at least 97%, at least 98%, at least 99% identity to the maturepart of the polypeptide of SEQ ID NO: 21 herein.

172. The enzyme composition of any of paragraphs 157-171, wherein theenzyme composition comprises a Bacillus licheniformis alpha-amylase anda Pyrococcus furiosus protease.

173. The enzyme composition of any of paragraphs 157-172, wherein theenzyme composition further comprises a glucoamylase.

174. The composition of any of paragraphs 157-173, wherein thePyrococcus furiosus is the one shown in SEQ ID NO: 13 herein.

175. The composition of any of paragraphs 157-174, wherein thePyrococcus furiosus protease is one having at least 80%, such as atleast 85%, such as at least 90%, such as at least 95%, such as at least96%, such as at least 97%, such as at least 98%, such as at least 99%identity to SEQ ID NO: 13 herein.

176. The composition of any of paragraphs 157-175, wherein the enzymecomposition further comprises a glucoamylase shown in SEQ ID NO: 14, ora variant thereof.

177. The composition of paragraph 152-166, wherein the glucoamylase hasa heat stability at 85° C., pH 5.3, of at least 20%, such as at least30%, preferably at least 35%.

178. The composition of any of paragraphs 176-177, wherein theglucoamylase has a relative activity pH optimum at pH 5.0 of at least90%, preferably at least 95%, preferably at least 97%.

179. The composition of any of paragraphs 176-178, wherein theglucoamylase has a pH stability at pH 5.0 of at least at least 80%, atleast 85%, at least 90%.

180. The composition of any of paragraphs 176-179, wherein theglucoamylase is derived from a strain of the genus Penicillium,especially a strain of Penicillium oxalicum disclosed as SEQ ID NO: 2 inWO 2011/127802 or SEQ ID NOs: 9 or 14 herein.

181. The composition of paragraph 176-170, wherein the glucoamylase hasat least 80%, more preferably at least 85%, more preferably at least90%, more preferably at least 91%, more preferably at least 92%, evenmore preferably at least 93%, most preferably at least 94%, and evenmost preferably at least 95%, such as even at least 96%, at least 97%,at least 98%, at least 99% or 100% identity to the mature polypeptideshown in SEQ ID NO: 2 in WO 2011/127802 or SEQ ID NOs: 9 or 14 herein.

182. The composition of any of paragraphs 176-181, wherein theglucoamylase is a variant of the Penicillium oxalicum glucoamylasedisclosed as SEQ ID NO: 2 in WO 2011/127802 or SEQ ID NO: 14 hereinhaving a K79V substitution (using the mature sequence shown in SEQ IDNO: 14 for numbering) such as a variant disclosed in WO 2013/053801.

183. The composition of any of paragraph 176-182, wherein thePenicillium oxalicum glucoamylase has a K79V substitution (using SEQ IDNO: 14 for numbering) and further one of the following:

T65A; or

Q327F; or

E501V; or

Y504T; or

Y504*; or

T65A+Q327F; or

T65A+E501V; or

T65A+Y504T; or

T65A+Y504*; or

Q327F+E501V; or

Q327F+Y504T; or

Q327F+Y504*; or

E501V+Y504T; or

E501V+Y504*; or

T65A+Q327F+E501V; or

T65A+Q327F+Y504T; or

T65A+E501V+Y504T; or

Q327F+E501V+Y504T; or

T65A+Q327F+Y504*; or

T65A+E501V+Y504*; or

Q327F+E501V+Y504*; or

T65A+Q327F+E501V+Y504T; or

T65A+Q327F+E501V+Y504*;

E501V+Y504T; or

T65A+K161S; or

T65A+Q405T; or

T65A+Q327W; or

T65A+Q327F; or

T65A+Q327Y; or

P11F+T65A+Q327F; or

R1K+D3W+K5Q+G7V+N8S+T10K+P11S+T65A+Q327F; or

P2N+P4S+P11F+T65A+Q327F; or

P11F+D26C+K33C+T65A+Q327F; or

P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; or

R1E+D3N+P4G+G6R+G7A+N8A+T10D+P11D+T65A+Q327F; or

P11F+T65A+Q327W; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or

P11F+T65A+Q327W+E501V+Y504T; or

T65A+Q327F+E501V+Y504T; or

T65A+S105P+Q327W; or

T65A+S105P+Q327F; or

T65A+Q327W+S364P; or

T65A+Q327F+S364P; or

T65A+S103N+Q327F; or

P2N+P4S+P11F+K34Y+T65A+Q327F; or

P2N+P4S+P11F+T65A+Q327F+D445N+V447S; or

P2N+P4S+P11F+T65A+I172V+Q327F; or

P2N+P4S+P11F+T65A+Q327F+N502*; or

P2N+P4S+P11F+T65A+Q327F+N502T+P563S+K571E; or

P2N+P4S+P11F+R31S+K33V+T65A+Q327F+N564D+K571S; or

P2N+P4S+P11F+T65A+Q327F+S377T; or

P2N+P4S+P11F+T65A+V325T+Q327W; or

P2N+P4S+P11F+T65A+Q327F+D445N+V447S+E501V+Y504T; or

P2N+P4S+P11F+T65A+I172V+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+S377T+E501V+Y504T; or

P2N+P4S+P11F+D26N+K34Y+T65A+Q327F; or

P2N+P4S+P11F+T65A+Q327F+I375A+E501V+Y504T; or

P2N+P4S+P11F+T65A+K218A+K221D+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+S103N+Q327F+E501V+Y504T; or

P2N+P4S+T10D+T65A+Q327F+E501V+Y504T; or

P2N+P4S+F12Y+T65A+Q327F+E501V+Y504T; or

K5A+P11F+T65A+Q327F+E501V+Y504T; or

P2N+P4S+T10E+E18N+T65A+Q327F+E501V+Y504T; or

P2N+T10E+E18N+T65A+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T568N; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+K524T+G526A; or

P2N+P4S+P11F+K34Y+T65A+Q327F+D445N+V447S+E501V+Y504T; or

P2N+P4S+P11F+R31S+K33V+T65A+Q327F+D445N+V447S+E501V+Y504T; or

P2N+P4S+P11F+D26N+K34Y+T65A+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+F80*+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+K112S+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T516P+K524T+G526A; or

P2N+P4S+P11F+T65A+Q327F+E501V+N502T+Y504*; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+S103N+Q327F+E501V+Y504T; or

K5A+P11F+T65A+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T516P+K524T+G526A; or

P2N+P4S+P11F+T65A+K79A+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+K79G+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+K791+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+K79L+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+K79S+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+L72V+Q327F+E501V+Y504T; or

S255N+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+E74N+V79K+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+G220N+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Y245N+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q253N+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+D279N+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+S359N+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+D370N+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+V460S+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+V460T+P468T+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+T463N+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+S465N+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+T477N+E501V+Y504T.

184. The composition of any of paragraphs 176-183, wherein theglucoamylase is the Penicillium oxalicum glucoamylase having a K79Vsubstitution (using SEQ ID NO: 14 for numbering) and further one of thefollowing substitutions:

-   -   P11F+T65A+Q327F    -   P2N+P4S+P11F+T65A+Q327F (using SEQ ID NO: 14 for numbering).

185. The composition of any of paragraphs 182-184, wherein theglucoamylase variant has at least 75% identity preferably at least 80%,more preferably at least 85%, more preferably at least 90%, morepreferably at least 91%, more preferably at least 92%, even morepreferably at least 93%, most preferably at least 94%, and even mostpreferably at least 95%, such as even at least 96%, at least 97%, atleast 98%, at least 99%, but less than 100% identity to the mature partof the polypeptide of SEQ ID NO: 14 herein.

186. The composition of any of paragraphs 157-185, further comprising apullulanase.

187. The composition of paragraph 186, wherein the pullulanase is afamily GH57 pullulanase, wherein the pullulanase preferably includes anX47 domain as disclosed in WO 2011/087836.

188. The composition of paragraphs 186-187, wherein the pullulanase isderived from a strain from the genus Thermococcus, includingThermococcus litoralis and Thermococcus hydrothermalis or a hybridthereof.

189. The composition of any of paragraphs 186-188, wherein thepullulanase is the truncated Thermococcus hydrothermalis pullulanase atsite X4 or a T. hydrothermalis/T. litoralis hybrid enzyme withtruncation site X4 disclosed in WO 2011/087836 or shown in SEQ ID NO: 12herein.

190. The composition of any of paragraphs 157-189 comprising

-   -   Bacillus stearothermophilus alpha-amylase, or a variant thereof;    -   Pyrococcus furiosus protease; and    -   Penicillium oxalicum glucoamylase,

wherein the ratio between alpha-amylase and protease is in the rangefrom 1:1 and 1:25 (micro gram alpha-amylase:micro gram protease).

191. The composition of any of paragraphs 157-190, comprising

-   -   an alpha-amylase, preferably derived from Bacillus        stearothermophilus, having a T½ (min) at pH 4.5, 85° C., 0.12 mM        CaCl₂) of at least 10;    -   Pyrococcus furiosus protease; and    -   Penicillium oxalicum glucoamylase,

wherein the ratio between alpha-amylase and protease is in the rangefrom 1:1 and 1:25 (micro gram alpha-amylase:micro gram protease).

192. The composition of any of paragraphs 157-191, comprising

-   -   an alpha-amylase derived from Bacillus stearothermophilus having        a double deletion I181+G182 and substitution N193F; and further        one of the following set of substitutions:        -   E129V+K177L+R179E;        -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;        -   V59A+Q89R+E129V+K177L+R179E+Q254S+M284V;        -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID NO:            1 herein for numbering);    -   Pyrococcus furiosus protease; and    -   Penicillium oxalicum glucoamylase in SEQ ID NO: 14 having        substitutions selected from the group of:    -   K79V;    -   K79V+P11F+T65A+Q327F; or    -   K79V+P2N+P4S+P11F+T65A+Q327F; or    -   K79V+P11F+D26C+K330+T65A+Q327F; or    -   K79V+P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; or    -   K79V+P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or    -   K79V+P11F+T65A+Q327W+E501V+Y504T (using SEQ ID NO: 14 for        numbering),

wherein the ratio between alpha-amylase and protease is in the rangefrom 1:1 and 1:25 (micro gram alpha-amylase:micro gram protease).

193. The enzyme composition of any of paragraphs 190-192, wherein theratio between alpha-amylase and protease is in the range between 1:1.2and 1:10, such as around 1:1.4 (micro gram alpha-amylase:micro gramprotease).

194. The enzyme composition of any of paragraphs 190-193, wherein theratio between alpha-amylase and glucoamylase is between 1:1 and 1:10,such as around 1:2 (micro gram alpha-amylase:micro gram glucoamylase).

The invention claimed is:
 1. A process of recovering oil from an ethanolproduction process comprising the steps of: a) liquefyingstarch-containing material at a temperature above the initialgelatinization temperature using: an alpha-amylase; and at least 2.5micro gram protease per gram dry solids (DS), wherein the protease isthe one shown in SEQ ID NO: 13 or a protease having at least 90%sequence identity thereto; b) saccharifying using a glucoamylase; c)fermenting using a fermenting organism; d) recovering the fermentationproduct to form whole stillage; e) separating the whole stillage intothin stillage and wet cake; f) optionally concentrating the thinstillage into syrup; wherein oil is recovered from the: liquefiedstarch-containing material after step a); and/or downstream fromfermentation step c).
 2. The process of claim 1, wherein oil isrecovered during, after, or both during and after liquefying thestarch-containing material; from the whole stillage; from the thinstillage; or from the syrup.
 3. The process of claim 1, wherein 2.5-100micro gram protease per gram DS are present or added in liquefactionstep a).
 4. The process of claim 1, wherein no nitrogen-compound isadded in during liquefaction step a), saccharification step b),fermentation step c), or simultaneous saccharification and fermentation(SSF).
 5. The process of claim 1, wherein the starch-containing materialis corn.
 6. The process of claim 1, wherein the protease is one havingat least 95% sequence identity to SEQ ID NO:
 13. 7. The process of claim1, wherein the protease has at least 97% sequence identity to SEQ ID NO:13.
 8. The process of claim 1, wherein the alpha-amylase is a Bacillusamylase.
 9. The process of claim 1, wherein the alpha-amylase is aBacillus stearothermophilus alpha-amylase.
 10. The process of claim 1,wherein the alpha-amylase is a Bacillus stearothermophilus alpha-amylasehaving a double deletion of positions I181+G182 or R179+G180 using SEQID NO: 1 for numbering.
 11. The process of claim 1, wherein thealpha-amylase has the amino acid sequence of SEQ ID NO: 1 or is avariant thereof having at least 85% sequence identity thereto.
 12. Theprocess of claim 1, wherein the alpha-amylase has at least 90% sequenceidentity to the amino acid sequence of SEQ ID NO:
 1. 13. The process ofclaim 1, wherein the alpha amylase has at least 95% sequence identity tothe amino acid sequence of SEQ ID NO:
 1. 14. The process of claim 1,further comprising using a glucoamylase in liquefying step (a).
 15. Theprocess of claim 1, further comprising using a glucoamylase inliquefying step (a), wherein the glucoamylase is a Penicillumglucoamylase.
 16. The process of claim 1, further comprising using aglucoamylase in liquefying step (a), wherein the glucoamylase has theamino acid sequence of SEQ ID NO: 14 or is a variant thereof having atleast 85% sequence identity thereto.
 17. The process of claim 16,wherein the glucoamylase has at least 90% sequence identity to the aminoacid sequence of SEQ ID NO:
 14. 18. The process of claim 16, wherein theglucoamylase has at least 95% sequence identity to the amino acidsequence of SEQ ID NO:
 14. 19. The process of claim 1, furthercomprising using a pullulanase in liquefying step (a).